Zsig33-like polynucleotides

ABSTRACT

The present invention relates to polynucleotide related to the zsig33 peptide, including agonists, antagonists, and antibodies. Methods of modulating gastric contractility, nutrient uptake, growth hormones, the secretion of digestive enzymes and hormones, and/or secretion of enzymes and/or hormones in the pancreas are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/921,371, filed Aug. 19, 2004, which is herein incorporated byreference, and which is a divisional of U.S. application Ser. No.09/853,253, filed May 10, 2001, now U.S. Pat. No. 6,897,286, which isherein incorporated by reference, and which claims the benefit of U.S.Provisional Application Ser. No. 60/203,300 filed on May 11, 2000.

BACKGROUND OF THE INVENTION

Many of the regulatory peptides that are important in maintainingnutritional homeostasis are found in the gastrointestinal environment.These peptides may be synthesized in the digestive system and actlocally, but can also be identified in the brain as well. In addition,the reverse is also found, i.e., peptides are synthesized in the brain,but found to regulate cells in the gastrointestinal tract. Thisphenomenon has been called the “brain-gut axis” and is important forsignaling satiety, regulating body temperature and other physiologicalprocesses that require feedback between the brain and gut.

The gut peptide hormones include gastrin, cholecystokinin (CCK),glucagon, secretin, gastric inhibitory peptide (GIP), vasoactiveintestinal polypeptide (VIP), motilin, somatostatin, pancreatic peptide(PP), substance P and neuropeptide Y (NPY), and use several differentmechanisms of action. For example, gastrin, motilin and CCK function asendocrine- and neurocrine-type hormones. Others, such as gastrin andGIP, are thought to act exclusively in an endocrine fashion. Other modesof action include a combination of endocrine, neurocrine and paracrineaction (somatostatin); exclusively neurocrine action (NPY); and acombination of neurocrine and paracrine actions (VIP and Substance P).Most of the gut hormone actions are mediated by membrane-bound receptorsand activate second messenger systems. For a review of gut peptides see,Mulvihill et al., in Basic and Clinical Endocrinology, pp. 551-570, 4thedition Greenspan F. S, and Baxter, J. D. editors., Appleton & Lange:Norwalk, Conn., 1994.

Many of these gut peptides are synthesized as inactive precursormolecules that require multiple peptide cleavages to be activated. Thefamily known as the “glucagon-secretin” family, which includes VIP,gastrin, secretin, motilin, glucagon and galanin, exemplifies peptidesregulated by multiple cleavages and post-translational modifications.

Motilin is a 22 amino acid peptide found in gut tissue of mammalianspecies (Domschke, W., Digestive Diseases 22(5):454-461, 1977). The DNAand amino acid sequences for porcine prepromotilin have been identified(U.S. Pat. No. 5,006,469). Motilin has been characterized as a factorcapable of increasing gastric motility, affecting the secretory functionof the stomach by stimulating pepsin secretion (Brown et al., CanadianJ. of Physiol. Pharmacol. 49:399-405, 1971), and recent evidencesuggests a role in myoelectric regulation of stomach and smallintestine. Cyclic increases of motilin have been correlated with phaseIII of the interdigestive myoelectric complex and the hunger contractionof the duodenum (Chey et al., in Gut Hormones, (eds.) Bloom, S. R., pp.355-358, Edinburgh, Churchill Livingstone, 1978; Lee et al, Am. J.Digestive Diseases, 23:789-795, 1978; and Itoh et al., Am. J. DigestiveDiseases, 23:929-935, 1978). Motilin and analogues of motilin have beendemonstrated to produce contraction of gastrointestinal smooth muscle,but not other types of smooth muscle cells (Strunz et al.,Gastroenterology 68:1485-1491, 1975).

The present invention is directed to a novel peptide fragment, and theDNA segment encoding it, of a previously described secreted protein,zsig33 (Sheppard, WO98/42840:1998). The present invention is alsodirected to a limited number of variants of said peptide fragment. Thediscovery of this novel peptide fragment is important for furtherelucidation of the how the body maintains its nutritional homeostasisand development of therapeutics to intervene in those processes, as wellas other uses that will be apparent from the teachings herein.

SUMMARY OF THE INVENTION

The present invention relates to novel Zsig33-like peptides, which areproduced by peptide cleavage from the C terminal peptide of zsig33.Within one aspect the invention provides an isolated polypeptideselected from the group consisting of: a polypeptide consisting of theamino acid sequence as shown in SEQ ID NOs:4, 5, or 6; a polypeptideconsisting of the amino acid sequence as shown in SEQ ID NOs:9, 10, or11; a polypeptide consisting the amino acid sequence as shown in SEQ IDNOs:14, 15, 16, or 17; a polypeptide consisting the amino acid sequenceas shown in SEQ ID NOs:20, 21, or 22; and a polypeptide consisting theamino acid sequence as shown in SEQ ID NOs:25, 26, or 27. Within anembodiment is provided an isolated polynucleotide encoding said isolatedpolypeptide. Within another embodiment the invention provides a methodof binding said isolated polypeptide. Within another embodiment, theinvention provides a method of modulating contractility ingastrointestinal tissue comprising applying the isolated polypeptide togastrointestinal tissue. Within another embodiment the inventionprovides a method of modulating pancreatic secretion of hormones anddigestive enzymes comprising administering the isolated polypeptide to amammal.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationof the second polypeptide or provide sites for attachment of the secondpolypeptide to a substrate. In principal, any peptide or protein forwhich an antibody or other specific binding agent is available can beused as an affinity tag. Affinity tags include a poly-histidine tract,protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:79524, 1985), substance P, Flag™ peptide (Hoppet al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide,maltose binding protein (Guan et al., Gene 67:21-30, 1987), cellulosebinding protein, thioredoxin, ubiquitin, T7 polymerase, or otherantigenic epitope or binding domain. See, in general, Ford et al.,Protein Expression and Purification 2: 95-107, 1991. DNAs encodingaffinity tags and other reagents are available from commercial suppliers(e.g., Pharmacia Biotech, Piscataway, N.J.; New England Biolabs,Beverly, Mass.; Eastman Kodak, New Haven, Conn.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “complement” of a polynucleotide molecule is a polynucleotide moleculehaving a complementary base sequence and reverse orientation as comparedto a reference sequence. For example, the sequence 5′ ATGCAC 3′ iscomplementary to 5′ GTGCAT 3′.

The term “corresponding to”, when applied to positions of amino acidresidues in sequences, means corresponding positions in a plurality ofsequences when the sequences are optimally aligned.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985)

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide or protein is substantially free of other polypeptides orproteins, particularly those of animal origin. It is preferred toprovide the polypeptides and proteins in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure. When usedin this context, the term “isolated” does not exclude the presence ofthe same polypeptide or protein in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

“Operably linked” means that two or more entities are joined togethersuch that they function in concert for their intended purposes. Whenreferring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

A “peptide-receptor complex” is formed when a peptide, or ligand, bindsto a receptor resulting in a change in the properties of the receptor.This change can result in an initiation of a cascade of reactionsleading to a change in cellular function, or the inability of thereceptor to bind additional peptides. The forming of a peptide-receptorcomplex can be reversible.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless. thus, a protein “consisting of”, for example, from 15 to1500 amino acid residues may further contain one or more carbohydratechains.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain or multi-peptide structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. Binding of ligand to receptorresults in a conformational change in the receptor that causes aninteraction between the effector domain and other molecule(s) in thecell. This interaction in turn leads to an alteration in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, increases in cyclic AMP production, mobilization ofcellular calcium, mobilization of membrane lipids, cell adhesion,hydrolysis of inositol lipids and hydrolysis of phospholipids. Ingeneral, receptors can be membrane bound, cytosolic or nuclear;monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergicreceptor) or multimeric (e.g., PDGF receptor, growth hormone receptor,IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptorand IL-6 receptor). A “secretory signal sequence” is a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

A “segment” is a portion of a larger molecule (e.g., polynucleotide orpolypeptide) having specified attributes. For example, a DNA segmentencoding a specified polypeptide is a portion of a longer DNA molecule,such as a plasmid or plasmid fragment, that, when read from the 5′ tothe 3′ direction, encodes the sequence of amino acids of the specifiedpolypeptide.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of novelpeptide fragments of a previously described secreted polypeptide knownas zsig33 (Sheppard, WO 98/42840). Zsig33 (shown in SEQ ID NOs: 1 and 2)which has homology to motilin has been found to be transcribed in thegastrointestinal system. The novel peptide fragments (shown in SEQ IDNOs:4-6, 9-11, 14-17, 20-22, and 25-26) have been designatedzsig-33-linker, zsig33-beta, zsig33-gamma, zsig33-delta andzsig33-epsilon peptides, herein referred to as zsig33-like peptides(ZS33LPs). Motilin is member of a family of polypeptides that regulatethe gastrointestinal physiology. The family of polypeptides important ingastrointestinal regulation to which motilin belongs includes glucagon,gastrin, galanin, and vasoactive intestinal peptide (VIP). Thesepolypeptides are synthesized in a precursor form that requires multiplesteps of processing to the active form. Particularly relevant to thepeptide of the present invention are motilin, glucagons, VIP andgalanin, where processing involves removal of signal sequence, followedby cleavage of one or more accessory peptides. In the case of glucagon,for example, multiple active peptides result form these cleavages. SeeDrucker, D., Pancreas 5:484-488, 1990. The resulting active peptides aregenerally small (10-30 amino acids) and may require furtherpost-translational modifications, such as amidation, sulfationacylation, octanoylation, or pyrrolidine carboxylic acid modification ofglutamine residues to form pyroglutamic acid.

Analysis of the tissue distribution of the mRNA corresponding to thezsig33 protein showed that expression is highest in stomach, followed byapparent but decreased expression levels in small intestine andpancreas. The EST for the secreted zsig33 protein is derived from apancreatic library, and has been shown in lung cDNA libraries. Thus, thenovel zsig33-beta and zsig33-gamma peptides would be expected tolocalize to these tissues or to any other tissues accessible by thecirculatory system of the body.

Many of the gut-brain peptides require multiple cleavages. For example,progastrin peptide is 101 amino acids, and is cleaved at the N-terminusresulting in sequentially smaller peptides (G34, G17 and G14) (Sugano etal., J. Biol. Chem. 260:11724-11729, 1985). Other peptides that requiremultiple processing steps include glucagon, for which C-terminalcleavages result in glucagon-like peptide 1 and glucagon-like peptide 2and galanin, in which processing involves cleavage of a C-terminalpeptide known as GMAP.

The ZS33LPs can modulate the absorption of glucose. Factors affectingthis modulation can include secretion of digestive enzymes or hormonesin organs and tissues involved in the gastrointestinal tract. An assayto measure the absorption of glucose is shown in Example 12.

Molecules of the present invention are related to additional cleavageproducts of the C terminal peptide which result from monobasic aminoacid cleavages at positions 51 (Arg), 75 (Arg), 85 (Lys) and 100 (Lys)of SEQ ID NO: 2. The resultant peptides are designated: zsig33-linkerpeptide, as shown in SEQ ID NOs:4-6; zsig33-beta peptide, as shown inSEQ ID NOs:9-11; zsig33-gamma peptide, as shown in SEQ ID NOs:14-17;zsig33-delta peptide, as shown in SEQ ID NOs:20-22; and zsig33-epsilonpeptide, as shown in SEQ ID NOs:25-26. Glucagon, another gut peptidehormone has similar post-translational processing. See Drucker, D.,Pancreas 5: 484-488, 1990. One skilled in the art will recognize thatsuch boundaries are approximate and may vary by +/−4 amino acids.

Polypeptides of the present invention comprise at least 6, preferably atleast 9, more preferably at least 15 contiguous amino acid residues ofSEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26. Within certainembodiments of the invention, the polypeptides comprise up to 25contiguous residues of SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26. Asdisclosed in more detail below, these polypeptides can further compriseadditional, non-ZS33LP polypeptide sequence(s).

Within the polypeptides of the present invention are polypeptides thatcomprise an epitope-bearing portion of a protein as shown in SEQ IDNOs:4-6, 9-11, 14-17, 20-22, and 25-26. An “epitope” is a region of aprotein to which an antibody can bind. See, for example, Geysen et al.,Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear orconformational, the latter being composed of discontinuous regions ofthe protein that form an epitope upon folding of the protein. Linearepitopes are generally at least 6 amino acid residues in length.Relatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, Sutcliffe et al., Science219:660-666, 1983. Antibodies that recognize short, linear epitopes areparticularly useful in analytic and diagnostic applications that employdenatured protein, such as Western blotting (Tobin, Proc. Natl. Acad.Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells ortissue samples. Antibodies to linear epitopes are also useful fordetecting fragments of ZS33LPs peptides, such as might occur in bodyfluids or cell culture media.

Antigenic, epitope-bearing polypeptides of the present invention areuseful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a ZS33LP. It is preferred that the amino acidsequence of the epitope-bearing polypeptide is selected to providesubstantial solubility in aqueous solvents, that is the sequenceincludes relatively hydrophilic residues, and hydrophobic residues aresubstantially avoided, and are described herein. Of interest within thepresent invention are polypeptides that comprise the entirezsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta andzsig33-epsilon polypeptides or portions thereof.

Polypeptides of the present invention can be prepared with one or moreamino acid substitutions, deletions or additions as compared to SEQ IDNOs:4-6, 9-11, 14-17, 20-22, and 25-26. These changes are preferably ofa minor nature, that is conservative amino acid substitutions and otherchanges that do not significantly affect the folding or activity of theprotein or polypeptide, and include amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, an amino orcarboxyl-terminal cysteine residue to facilitate subsequent linking tomaleimide-activated keyhole limpet hemocyanin, a small linker peptide ofup to about 20-25 residues, or an extension that facilitatespurification (an affinity tag) as disclosed above. Two or more affinitytags may be used in combination. Polypeptides comprising affinity tagscan further comprise a polypeptide linker and/or a proteolytic cleavagesite between the zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta,and zsig33-epsilon polypeptide and the affinity tag. Preferred cleavagesites include thrombin cleavage sites and factor Xa cleavage sites.

Auxiliary domains can be fused to ZS33LP polypeptides to target them tospecific cells, tissues, or macromolecules (e.g., collagen). Forexample, a ZS33LP or protein can be targeted to a predetermined celltype by fusing a zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta,or zsig33-epsilon polypeptide to a ligand that specifically binds to areceptor on the surface of the target cell. In this way, polypeptidesand proteins can be targeted for therapeutic or diagnostic purposes. Azsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, orzsig33-epsilon polypeptide can be fused to two or more moieties, such asan affinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9,1996.

As disclosed above, the polypeptides of the present invention compriseat least nine contiguous residues of SEQ ID NOs:4-6, 9-11, 14-17, 20-22,and 25-26. These polypeptides may further comprise additional residuesas shown in SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26, a variant ofSEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26 or another protein asdisclosed herein. When variants of SEQ ID NOs:4-6, 9-11, 14-17, 20-22,and 25-26 are employed, the resulting polypeptide will preferably be atleast 80%, more preferably at least 90% or 95% identical to thecorresponding region of SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26.Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 1 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:

$\frac{\left( {{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}} \right)}{\left\lbrack \begin{matrix}{{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}}\mspace{14mu}} \\{{the}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}} \\{\mspace{14mu} {{sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}}\end{matrix} \right\rbrack} \times 100$

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

The level of identity between amino acid sequences can be determinedusing the “FASTA” similarity search algorithm disclosed by Pearson andLipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth.Enzymol. 183:63, 1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26) and a test sequence thathave either the highest density of identities (if the ktup variableis 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, 1990 (ibid.).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

The present invention includes polypeptides having one or moreconservative amino acid changes as compared with the amino acid sequenceof SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26. The BLOSUM62 matrix(Table 1) is an amino acid substitution matrix derived from about 2,000local multiple alignments of protein sequence segments, representinghighly conserved regions of more than 500 groups of related proteins(Henikoff and Henikoff, ibid.). Thus, the BLOSUM62 substitutionfrequencies can be used to define conservative amino acid substitutionsthat may be introduced into the amino acid sequences of the presentinvention. As used herein, the term “conservative amino acidsubstitution” refers to a substitution represented by a BLOSUM62 valueof greater than −1. For example, an amino acid substitution isconservative if the substitution is characterized by a BLOSUM62 value of0, 1, 2, or 3; Preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3),while more preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993). Similarly, the d-stereoisomer of the amino acidscan also be substituted.

Amino acid sequence changes are made in ZS33LP polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. Amino acid residues that are within regions or domains thatare critical to maintaining structural integrity can be determined.Within these regions one can identify specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing, and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,determination of structure will be accompanied by evaluation of activityof modified molecules. The effects of amino acid sequence changes can bepredicted by, for example, computer modeling using available software(e.g., the Insight II® viewer and homology modeling tools; MSI, SanDiego, Calif.) or determined by analysis of crystal structure (see,e.g., Lapthorn et al, Nature 369:455-461, 1994; Lapthorn et al., Nat.Struct. Biol. 2:266-268, 1995). Protein folding can be measured bycircular dichroism (CD). Measuring and comparing the CD spectragenerated by a modified molecule and standard molecule are routine inthe art (Johnson, Proteins 7:205-214, 1990). Crystallography is anotherwell known and accepted method for analyzing folding and structure.Nuclear magnetic resonance (NMR), digestive peptide mapping and epitopemapping are other known methods for analyzing folding and structuralsimilarities between proteins and polypeptides (Schaanan et al., Science257:961-964, 1992). Mass spectrometry and chemical modification usingreduction and alkylation can be used to identify cysteine residues thatare associated with disulfide bonds or are free of such associations(Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci.2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732,1994). Alterations in disulfide bonding will be expected to affectprotein folding. These techniques can be employed individually or incombination to analyze and compare the structural features that affectfolding of a variant protein or polypeptide to a standard molecule todetermine whether such modifications would be significant.

Hydrophilicity profiles of SEQ ID NOs:4, 9 and 14 have been performed.Particularly hydrophilic regions of these sequences are from residue 7to residue 18 of SEQ ID NO:4, residue 14 to residue 21 of SEQ ID NO:9,and residue 7 to residue 16 of SEQ ID NO:14. Those skilled in the artwill recognize that this hydrophilicity will be taken into account whendesigning alterations in the amino acid sequence of a zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilonpolypeptides, respectively, so as not to disrupt the overall profile.

Essential amino acids in the polypeptides of the present invention canbe identified experimentally according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed ZS33LP DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994. Briefly, variant genes are generated by in vitrohomologous recombination by random fragmentation of a parent genefollowed by reassembly using PCR, resulting in randomly introduced pointmutations. This technique can be modified by using a family of parentgenes, such as allelic variants or genes from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

In many cases, the structure of the final polypeptide product willresult from processing of the nascent polypeptide chain by the hostcell, thus the final sequence of a ZS33LP produced by a host cell willnot always correspond to the full sequence encoded by the expressedpolynucleotide. For example, expressing the complete zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilon sequence ina cultured mammalian cell is expected to result in removal of at leastthe secretory peptide, while the same polypeptide produced in aprokaryotic host would not be expected to be cleaved. Differentialprocessing of individual chains may result in heterogeneity of expressedpolypeptides.

Zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon proteins of the present invention are characterized bytheir activity, that is, modulation of gastrointestinal contractility,modulation of gastric motility, modulation of glucose uptake, modulationof insulin secretion, modulation of secretion of enzymes and/or hormonesin the pancreas, or binding a ZS33LP binding partner. Biologicalactivity of zsig33-linker, zsig33-beta, zsig33-gamma, and zsig33-delta,and zsig33-epsilon proteins is assayed using in vitro or in vivo assaysdesigned to detect gastric emptying, contractility, glucose absorption,or binding proliferation, differentiation, migration or adhesion; orchanges in cellular metabolism. Many suitable assays are known in theart, and representative assays are disclosed herein. Assays usingcultured cells are most convenient for screening, such as fordetermining the effects of amino acid substitutions, deletions, orinsertions. Assays can be performed using exogenously produced proteins,or may be carried out in vivo or in vitro using cells expressing thepolypeptide(s) of interest. Assays can be conducted using zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilon proteinsalone or in combination with other growth factors, such as members ofthe VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stemcell factor). Representative assays are disclosed below.

Mutagenesis methods as disclosed above can be combined with high volumeor high-throughput screening methods to detect biological activity ofZS33LP variant polypeptides. Assays that can be scaled up for highthroughput include mitogenesis assays, which can be run in a 96-wellformat. Mutagenized DNA molecules that encode active zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilon polypeptidescan be recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptide fragments or variants of SEQ IDNOs:4-6, 9-11, 14-17, 20-22, and 25-26 that retain the activity ofwild-type zsig33-linker, zsig33-beta, zsig-33 gamma, zsig33-delta, andzsig33-epsilon.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, and zsig33-epsilon polypeptides disclosedabove. Representative DNA sequences encoding the amino acid sequences ofSEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26 are shown in SEQ ID NOs:3,8, 13, 19, and 24, respectively. Those skilled in the art will readilyrecognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. SEQ ID NO:7 is a degenerate DNA sequence that encompasses allDNAs that encode the zsig33-linker peptide. SEQ ID NO:12 is a degenerateDNA sequence that encompasses all DNAs that encode the zsig33-betapeptide. SEQ ID NO:18 is a degenerate DNA sequence that encompasses allDNAs that encode the zsig33-gamma peptide. SEQ ID NO:23 is a degenerateDNA sequence that encompasses all DNAs that encode the zsig33-deltapeptide. SEQ ID NO:27 is a degenerate DNA sequence that encompasses allDNAs that encode the zsig33-epsilon peptide. Those skilled in the artwill recognize that the degenerate sequences of SEQ ID NOs:7, 12, 18, 23and 27 also provide all RNA sequences encoding SEQ ID NOs:4, 9, 14, 20and 25, respectively, by substituting U for T. Thus, zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig-33 epsilonpolypeptide-encoding polynucleotides comprising nucleotides 1 to 72 ofSEQ ID NO:3, or nucleotides 1 to 75 of SEQ ID NO:8, nucleotides 1 to 51of SEQ ID NO:13, nucleotides 1 to 30 of SEQ ID NO:19, or nucleotide 1 to45 of SEQ ID NO:24 and their RNA equivalents are contemplated by thepresent invention, as are segments of SEQ ID NOs:7, 12, 18, 23, and 27encoding other zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta,and zsig33-epsilon polypeptides disclosed herein. Table 2 sets forth theone-letter codes used within SEQ ID NOs:7, 12, 18, 23, and 27 to denotedegenerate nucleotide positions. “Resolutions” are the nucleotidesdenoted by a code letter. “Complement” indicates the code for thecomplementary nucleotide(s). For example, the code Y denotes either C orT, and its complement R denotes A or G, A being complementary to T, andG being complementary to C.

TABLE 2 Nucleotide Resolutions Complement Resolutions A A T T C C G G GG C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G SC|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|TH A|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs:7, 12, 18, 23, and 27encompassing all possible codons for a given amino acid, are set forthin Table 3, below.

TABLE 3 Amino One-Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAG TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap — —

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NOs:4-6, 9-11, 14-17, 20-22, and 25-26. Variantsequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit preferential codon usage. See, in general, Granthamet al., Nuc. Acids Res. 8:1893-912, 1980; Haas et al. Curr. Biol.6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjean andFiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986;and Ikemura, J. Mol. Biol. 158:573-97, 1982. Introduction of preferredcodon sequences into recombinant DNA can, for example, enhanceproduction of the protein by making protein translation more efficientwithin a particular cell type or species. Therefore, the degeneratecodon sequences disclosed in SEQ ID NOs:7, 12, 18, 23, and 27 serve astemplates for optimizing expression of polynucleotides in various celltypes and species commonly used in the art and disclosed herein.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NOs:3,8, 13, 19, and 24, or a sequence complementary thereto under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is up toabout 0.03 M at pH 7 and the temperature is at least about 60° C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of ZS33LP RNA. Stomach cells are preferred.Pancreas is another preferred source. Total RNA can be prepared usingguanidine HCl extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. Polynucleotides encoding zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilon polypeptidesare then identified and isolated by, for example, hybridization or PCR.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS:1 and 2 represent a single allele of human zsig33. Allelicvariants of these sequences can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (“orthologs”). Of particular interestare ZS33LP polypeptides from other mammalian species, including murine,porcine, ovine, bovine, canine, feline, equine, and other primatepolypeptides. Orthologs of human zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, and zsig33-epsilon can be cloned usinginformation and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses ZS33LP as disclosed above. A library is then prepared frommRNA of a positive tissue or cell line. A zsig33-linker, zsig33-beta,zsig-33 gamma, zsig33-delta, and zsig33-epsilon-encoding cDNA can thenbe isolated by a variety of methods, such as by probing with a completeor partial human cDNA or with one or more sets of degenerate probesbased on the disclosed sequence. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human zsig33-linker,zsig33-beta, zsig33-gamma, zsig33-delta, and zsig33-epsilon sequencesdisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, and zsig33-epsilon polypeptides. Similartechniques can also be applied to the isolation of genomic clones.

For any ZS33LP polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 2 and 3, above. Moreover, those of skill in the art canuse standard software to devise zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, and zsig33-epsilon variants based upon thenucleotide and amino acid sequences described herein. The presentinvention thus provides a computer-readable medium encoded with a datastructure that provides at least one of the following sequences: SEQ IDNO:1 to SEQ ID NO:28, and portions thereof. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP™ disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

The zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon polypeptides of the present invention, includingbiologically active fragments, and fusion polypeptides can be producedaccording to conventional techniques using cells into which have beenintroduced an expression vector encoding the polypeptide. As usedherein, “cells into which have been introduced an expression vector”include both cells that have been directly manipulated by theintroduction of exogenous DNA molecules and progeny thereof that containthe introduced DNA. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,NY, 1987.

In general, a DNA sequence encoding a zsig33-linker, zsig33-beta,zsig33-gamma or zsig33-delta, and zsig33-epsilon polypeptide is operablylinked to other genetic elements required for expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zsig33-linker, zsig33-beta, zsig33-gamma and zsig33-delta,and zsig33-epsilon polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of human zsig33 (i.e., fromresidue 1 to residue 23 of SEQ ID NO:2, or may be derived from anothersecreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655) orsynthesized de novo. The secretory signal sequence is operably linked tothe zsig33-linker, zsig33-beta, zsig33-gamma or zsig33-delta, andzsig33-epsilon DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter. Expression vectors for use in mammaliancells include pZP-1 and pZP-9, which have been deposited with theAmerican Type Culture Collection, Manassas, Va. USA under accessionnumbers 98669 and 98668, respectively, and derivatives thereof.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

The adenovirus system (disclosed in more detail below) can also be usedfor protein production in vitro. By culturing adenovirus-infectednon-293 cells under conditions where the cells are not rapidly dividing,the cells can produce proteins for extended periods of time. Forinstance, BHK cells are grown to confluence in cell factories, thenexposed to the adenoviral vector encoding the secreted protein ofinterest. The cells are then grown under serum-free conditions, whichallows infected cells to survive for several weeks without significantcell division. In an alternative method, adenovirus vector-infected 293cells can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(See Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant, lysate, or membranefractions depending on the disposition of the expressed protein in thecell. Within the infected 293 cell production protocol, non-secretedproteins can also be effectively obtained.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV)according to methods known in the art. Within a preferred method,recombinant baculovirus is produced through the use of atransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Bac™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing azsig33-linker, zsig33-beta, zsig-33 gamma, zsig33-delta, andzsig33-epsilon-encoding sequence is transformed into E. coli host cells,and the cells are screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.Recombinant virus that expresses zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, and zsig33-epsilon protein is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HighFive™ cells; Invitrogen, Carlsbad, Calif.). See, for example, U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally known in the art.

Other higher eukaryotic cells can also be used as hosts, including plantcells and avian cells. The use of Agrobacterium rhizogenes as a vectorfor expressing genes in plant cells has been reviewed by Sinkar et al.,J. Biosci. (Bangalore) 11:47-58, 1987.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kaisaki, U.S. Pat. No. 4,599,311; Kaisaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kaisakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kaisaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat.No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;5,139,936 and 4,661,454. Transformation systems for other yeasts,including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondii and Candida maltosa are known in theart. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465,1986; Cregg, U.S. Pat. No. 4,882,279; and Raymond et al., Yeast 14,11-23, 1998. Aspergillus cells may be utilized according to the methodsof McKnight et al., U.S. Pat. No. 4,935,349. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteinsin Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808,5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing azsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon polypeptide in bacteria such as E. coli, the polypeptidemay be retained in the cytoplasm, typically as insoluble granules, ormay be directed to the periplasmic space by a bacterial secretionsequence. In the former case, the cells are lysed, and the granules arerecovered and denatured using, for example, guanidine isothiocyanate orurea. The denatured polypeptide can then be refolded and dimerized bydiluting the denaturant, such as by dialysis against a solution of ureaand a combination of reduced and oxidized glutathione, followed bydialysis against a buffered saline solution. In the latter case, thepolypeptide can be recovered from the periplasmic space in a soluble andfunctional form by disrupting the cells (by, for example, sonication orosmotic shock) to release the contents of the periplasmic space andrecovering the protein, thereby obviating the need for denaturation andrefolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

It is preferred to purify the polypeptides and proteins of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant zsig33-linker, zsig33-beta, zsig33-gamma,zsig33-delta, and zsig33-epsilon proteins (including chimericpolypeptides and multimeric proteins) are purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al., ibid.Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

Zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. In general, see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al.,Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co.,Rockford, Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; andAtherton et al., Solid Phase Peptide Synthesis: A Practical Approach,IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageousfor the preparation of smaller polypeptides.

ZS33LPs may be monomers or multimers; glycosylated or non-glycosylated;pegylated or non-pegylated; amidated or non-amidated; sulfated ornon-sulfated; and may or may not include an initial methionine aminoacid residue. For example, ZS33LPs can also be synthesized by exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. The peptides arepreferably prepared by solid phase peptide synthesis, for example asdescribed by Merrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesisis carried out with amino acids that are protected at the alpha-aminoterminus. Trifunctional amino acids with labile side-chains are alsoprotected with suitable groups to prevent undesired chemical reactionsfrom occurring during the assembly of the polypeptides. The alpha-aminoprotecting group is selectively removed to allow subsequent reaction totake place at the amino-terminus. The conditions for the removal of thealpha-amino protecting group do not remove the side-chain protectinggroups.

The alpha-amino protecting groups are those known to be useful in theart of stepwise polypeptide synthesis. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aryl type protectinggroups (e.g., biotinyl), aromatic urethane type protecting groups [e.g.,benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

In tBoc chemistry, the preferred side-chain protecting groups are tosylfor arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

For the synthesis of phosphopeptides, either direct or post-assemblyincorporation of the phosphate group is used. In the directincorporation strategy, the phosphate group on serine, threonine ortyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

Solid phase synthesis is usually carried out from the carboxyl-terminusby coupling the alpha-amino protected (side-chain protected) amino acidto a suitable solid support. An ester linkage is formed when theattachment is made to a chloromethyl, chlortrityl or hydroxymethylresin, and the resulting polypeptide will have a free carboxyl group atthe C-terminus. Alternatively, when an amide resin such asbenzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry)and Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bondis formed and the resulting polypeptide will have a carboxamide group atthe C-terminus. These resins, whether polystyrene- or polyamide-based orpolyethyleneglycol-grafted, with or without a handle or linker, with orwithout the first amino acid attached, are commercially available, andtheir preparations have been described by Stewart et al., “Solid PhasePeptide Synthesis” (2nd Edition), (Pierce Chemical Co., Rockford, Ill.,1984) and Bayer & Rapp Chem. Pept. Prot. 3:3 (1986); and Atherton etal., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,Oxford, 1989.

The C-terminal amino acid, protected at the side chain if necessary, andat the alpha-amino group, is attached to a hydroxylmethyl resin usingvarious activating agents including dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). Itcan be attached to chloromethyl or chlorotrityl resin directly in itscesium tetramethylammonium salt form or in the presence of triethylamine(TEA) or diisopropylethylamine (DIEA). First amino acid attachment to anamide resin is the same as amide bond formation during couplingreactions.

Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH2Cl2 or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970.

After the entire assembly of the desired peptide, the peptide-resin iscleaved with a reagent with proper scavengers. The Fmoc peptides areusually cleaved and deprotected by TFA with scavengers (e.g., H2O,ethanedithiol, phenol and thioanisole). The tBoc peptides are usuallycleaved and deprotected with liquid HF for 1-2 hours at −5 to 0° C.,which cleaves the polypeptide from the resin and removes most of theside-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury (II) acetate and alternatively by iodine, thallium (III)trifluoroacetate or silver tetrafluoroborate which simultaneouslyoxidize cysteine to cystine. Other strong acids used for tBoc peptidecleavage and deprotection include trifluoromethanesulfonic acid (TFMSA)and trimethylsilyltrifluoroacetate (TMSOTf).

The activity of molecules of the present invention can be measured usinga variety of assays that measure for example, modulation ofgastrointestinal contractility, modulation of gastric motility,modulation of glucose uptake, modulation of insulin secretion,modulation of secretion of enzymes and/or hormones in the pancreas, orbinding a ZS33LP binding partner. Of particular interest are changes incontractility of smooth muscle cells. For example, the contractileresponse of segments of mammalian duodenum or other gastrointestinalsmooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility1:150-159, 1989, incorporated herein by reference). An exemplary in vivoassay uses an ultrasonic micrometer to measure the dimensional changesradially between commissures and longiturdinally to the plane of thevalve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390,1995).

Gastric motility is generally measured in the clinical setting as thetime required for gastric emptying and subsequent transit time throughthe gastrointestinal tract. Gastric emptying scans are well known tothose skilled in the art, and briefly, comprise use of an oral contrastagent, such as barium, or a radiolabeled meal. Solids and liquids can bemeasured independently. A test food or liquid is radiolabeled with anisotope (e.g. ^(99m)Tc), and after ingestion or administration, transittime through the gastrointestinal tract and gastric emptying aremeasured by visualization using gamma cameras (Meyer et al., Am. J. Dig.Dis. 21:296, 1976; Collins et al., Gut 24:1117, 1983; Maughan et al.,Diabet. Med. 13 9 Supp. 5:S6-10, 1996 and Horowitz et al., Arch. Intern.Med. 145:1467-1472, 1985). These studies may be performed before andafter the administration of a promotility agent to quantify the efficacyof the drug.

In view of the tissue distribution observed for zsig33, agonists(including the natural ligand/substrate/cofactor/synthetic and naturallyoccurring peptides, and variants, etc.) and antagonists have enormouspotential in both in vitro and in vivo applications. Compoundsidentified as ZS33LP agonists are useful for modulation ofgastrointestinal contractility, modulation of gastric motility,modulation of glucose uptake, modulation of insulin secretion,modulation of secretion of enzymes and/or hormones in the pancreas, orbinding a ZS33LP binding partner in vivo and in vitro. For example,agonist compounds are useful as components of defined cell culture mediaand regulate the uptake of nutrients, and thus are useful inspecifically promoting the growth and/or development of gastrointestinalcells such as G cells, enterochromaffin cells and the epithelial mucosaof the stomach, duodenum, proximal jejunum, antrum and fundus.Additionally, ZS33LP polypeptides and ZS33LP agonists are useful as aresearch reagent, such as for the expansion, differentiation, growthfactor and hormone secretion, and/or cell-cell interactions of tissuesassociated with the gastrointestinal system, brain, and/or centralnervous system.

The family of gut-brain peptides has been associated with neurologicaland CNS functions. For example, NPY, a peptide with receptors in boththe brain and the gut has been shown to stimulate appetite whenadministered to the central nervous system (Gehlert, Life Sciences55(6):551-562, 1994). Motilin immunoreactivity has been identified indifferent regions of the brain, particularly the cerebellum, and in thepituitary (Gasparini et al., Hum. Genetics 94(6):671-674, 1994). Motilinhas been found to coexist with neurotransmitter γ-aminobutyric acid incerebellum (Chan-Patay, Proc. Sym. 50th Anniv. Meet. Br. Pharmalog.Soc.:1-24, 1982). Physiological studies have provided some evidence thatmotilin has an affect on feeding behavior (Rosenfield et al., Phys.Behav. 39(6):735-736, 1987), bladder control, pituitary growth hormonerelease. Other gut-brain peptides, such as CCK, enkephalin, VIP andsecretin have been shown to be involved in control of blood pressure,heart rate, behavior, and pain modulation, in addition to be active inthe digestive system. Therefore, ZS33LPs, could be expected to have someneurological association.

Additionally, other members of the gut-brain peptides, such as CCK,gastrin, and the like, have been shown to modulate secretion ofpancreatic enzymes and hormones. Thus, zsig33-beta and zsig33-gammapeptides can be used to modulate secretion of pancreatic enzymes andhormones.

Similarly, other members of this family are known to modulate thesecretion of endogenous proteins, such as the manner in which glucagonmodulates the secretion of insulin. ZS33LPs can be used to modulate thesecretion of non-ZS33LP proteins such as, for example, GLP-1, growthhormone, somatostatin, and the like. One advantage of growth hormonesecretagogues, in general, is their ability to amplify endogenouspulsatile growth hormone secretion while maintaining normal feedbackmechanisms. Another important effect is the ability to restore seruminsulin-like growth factor-1 (IGF-1) levels in elderly adults toconcentrations similar to those of young adults. See Hansen, B. S. etal., Eur. J. Endocrinol. 141:180-189, 1999. Thus, ZS33LPs may be usefulfor modulating secretion of growth hormone and insulin-like growthfactor 1.

Using site-specific changes in the amino acid and DNA sequences of thepresent invention analogs can be made that are either antagonists,agonists or partial agonists (Macielag et al., Peptides: Chem. Struct.Biol. pp. 659, 1996). Antagonists are useful for clinical conditionsassociated with gastrointestinal hypermotility such as diarrhea andCrohn's disease. Antagonists are also useful as research reagents forcharacterizing sites of ligand-receptor interaction.

Target cells for use in zsig33-linker, zsig33-beta, zsig33-gamma,zsig33-delta, and zsig33-epsilon activity assays include, withoutlimitation, gastrointestinal cells (especially smooth muscle cells),pancreas cells (islets and beta cells), and pituitary cells. Endothelialcells and hematopoietic cells are derived from a common ancestral cell,the hemangioblast (Choi et al., Development 125:725-732, 1998).

Activity of ZS33LP proteins can be measured in vitro using culturedcells or in vivo by administering molecules of the claimed invention toan appropriate animal model. Assays measuring cell proliferation ordifferentiation are well known in the art. For example, assays measuringproliferation include such assays as chemosensitivity to neutral red dye(Cavanaugh et al., Investigational New Drugs 8:347-354, 1990),incorporation of radiolabelled nucleotides (as disclosed by, e.g.,Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol.Cell. Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem.179:1-7, 1989), incorporation of 5-bromo-2′-deoxyuridine (BrdU) in theDNA of proliferating cells (Porstmann et al., J. Immunol. Methods82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988). Differentiation can be assayed using suitableprecursor cells that can be induced to differentiate into a more maturephenotype. Assays measuring differentiation include, for example,measuring cell-surface markers associated with stage-specific expressionof a tissue, enzymatic activity, functional activity or morphologicalchanges (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,161-171, 1989; all incorporated herein by reference).

Zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon activity may also be detected using assays designed tomeasure zsig33-linker, zsig33-beta, zsig-33 gamma, zsig33-delta, andzsig33-epsilon-induced production of one or more additional growthfactors or other macromolecules. Preferred such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules produced by the liver. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art.

Cell migration is assayed essentially as disclosed by Kähler et al.(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. A typical assay is performed using modified Boydenchambers with a polystryrene membrane separating the two chambers(Transwell; Corning Costar Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve. Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997).

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle ishing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

The activity of ZS33LP proteins can be measured with a silicon-basedbiosensor microphysiometer that measures the extracellular acidificationrate or proton excretion associated with receptor binding and subsequentphysiologic cellular responses. An exemplary such device is theCytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including ZS33LPs, their agonists, and antagonists. Preferably,the microphysiometer is used to measure responses of a ZS33LP-responsiveeukaryotic cell, compared to a control eukaryotic cell that does notrespond to ZS33LP. ZS33LP-responsive eukaryotic cells comprise cellsinto which a receptor for a ZS33LP has been transfected, therebycreating a cell that is responsive to a ZS33LP as well as cellsnaturally responsive to a ZS33LP. Differences, measured by a change inextracellular acidification in the response of cells exposed to a ZS33LPpolypeptide, relative to a control not exposed to a ZS33LP, are a directmeasurement of ZS33LP modulated cellular responses. Moreover, suchZS33LP-modulated responses can be assayed under a variety of stimuli.The present invention thus provides methods of identifying agonists andantagonists of ZS33LP proteins, comprising providing cells responsive toa ZS33LP polypeptide, culturing a first portion of the cells in theabsence of a test compound, culturing a second portion of the cells inthe presence of a test compound, and detecting a change, for example, anincrease or diminution, in a cellular response of the second portion ofthe cells as compared to the first portion of the cells. The change incellular response is shown as a measurable change in extracellularacidification rate. Culturing a third portion of the cells in thepresence of a ZS33LP protein and the absence of a test compound providesa positive control for the ZS33LP-responsive cells and a control tocompare the agonist activity of a test compound with that of the ZS33LPpolypeptide. Antagonists of ZS33LPs can be identified by exposing thecells to ZS33LPs protein in the presence and absence of the testcompound, whereby a reduction in ZS33LP-stimulated activity isindicative of antagonist activity in the test compound.

Expression of ZS33LP polynucleotides in animals provides models forfurther study of the biological effects of overproduction or inhibitionof protein activity in vivo. ZS33LP-encoding polynucleotides andantisense polynucleotides can be introduced into test animals, such asmice, using viral vectors or naked DNA, or transgenic animals can beproduced.

One in vivo approach for assaying proteins of the present inventionutilizes viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene is deleted from the viral vector, and the virus willnot replicate unless the E1 gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

An alternative method of gene delivery comprises removing cells from thebody and introducing a vector into the cells as a naked DNA plasmid. Thetransformed cells are then re-implanted in the body. Naked DNA vectorsare introduced into host cells by methods known in the art, includingtransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem.263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; andJohnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

Transgenic mice, engineered to express a zsig33-linker, zsig33-beta,zsig33-gamma, zsig33-delta, or zsig33-epsilon gene, and mice thatexhibit a complete absence of zsig33-linker, zsig33-beta, zsig33-gamma,zsig33-delta, or zsig33-epsilon gene function, referred to as “knockoutmice” (Snouwaert et al., Science 257:1083, 1992), can also be generated(Lowell et al., Nature 366:740-742, 1993). These mice can be employed tostudy the ZS33LP gene of interest and the protein encoded thereby in anin vivo system. Transgenic mice are particularly useful forinvestigating the role of ZS33LP proteins in early development in thatthey allow the identification of developmental abnormalities or blocksresulting from the over- or underexpression of a specific factor. Seealso, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science277:48-50, 1997. Preferred promoters for transgenic expression includepromoters from metallothionein and albumin genes.

Antisense methodology can be used to inhibit ZS33LP gene transcriptionto examine the effects of such inhibition in vivo. Polynucleotides thatare complementary to a segment of a zsig33-linker, zsig33-beta, zsig-33gamma, zsig33-delta, or zsig33-epsilon-encoding polynucleotides (e.g., apolynucleotide as set forth in SEQ ID NOs:3, 8, 13, 19, and 24) aredesigned to bind to zsig33-linker, zsig33-beta, zsig-33 gamma,zsig33-delta, or zsig33-epsilon-encoding mRNA and to inhibit translationof such mRNA. Such antisense oligonucleotides can also be used toinhibit expression of ZS33LP polypeptide-encoding genes in cell culture.

For pharmaceutical use, zsig33-linker, zsig33-beta, zsig33-gammazsig33-delta, or zsig33-epsilon proteins are formulated for topical orparenteral, particularly intravenous or subcutaneous, delivery accordingto conventional methods. In general, pharmaceutical formulations willinclude a zsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, orzsig33-epsilon zsig33-delta, or zsig33-epsilon zsig33-delta, orzsig33-epsilon polypeptide in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. ZS33LPs will preferably be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. Dosing is daily or intermittentlyover the period of treatment. Intravenous administration will be bybolus injection or infusion over a typical period of one to severalhours. Sustained release formulations can also be employed. In general,a therapeutically effective amount of ZS33LP is an amount sufficient toproduce a clinically significant change in the treated condition, suchas a clinically significant change in hematopoietic or immune function,a significant reduction in morbidity, or a significantly increasedhistological score.

ZS33LP proteins, agonists, and antagonists are useful for modulating theexpansion, proliferation, activation, differentiation, migration, ormetabolism of responsive cell types, which include both primary cellsand cultured cell lines. Of particular interest in this regard aregastrointestinal smooth muscle cells, pancreas cells and pituitarycells. ZS33LP polypeptides are added to tissue culture media for thesecell types at a concentration of about 10 pg/ml to about 100 ng/ml.Those skilled in the art will recognize that ZS33LP proteins can beadvantageously combined with other growth factors in culture media.

Within the laboratory research field, ZS33LP proteins can also be usedas molecular weight standards or as reagents in assays for determiningcirculating levels of the protein, such as in the diagnosis of disorderscharacterized by over- or under-production of ZS33LP proteins or in theanalysis of cell phenotype.

ZS33LP proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of a ZS33LP protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of ZS33LP activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofZS33LP-dependent cellular responses. For example, ZS33LP-responsive celllines can be transfected with a reporter gene construct that isresponsive to a ZS33LP-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and generally comprise aresponse element operably linked to a gene encoding an assay detectableprotein, such as luciferase. DNA response elements can include, but arenot limited to, cyclic AMP response elements (CRE), hormone responseelements (HRE) insulin response element (IRE) (Nasrin et al., Proc.Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE)(Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements arereviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 andHabener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone responseelements are reviewed in Beato, Cell 56:335-44; 1989. Candidatecompounds, solutions, mixtures or extracts are tested for the ability toinhibit the activity of a ZS33LP on the target cells as evidenced by adecrease in ZS33LP stimulation of reporter gene expression. Assays ofthis type will detect compounds that directly block ZS33LP binding tocell-surface receptors, as well as compounds that block processes in thecellular pathway subsequent to receptor-ligand binding. In thealternative, compounds or other samples can be tested for directblocking of ZS33LP binding to receptor using ZS33LP tagged with adetectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, andthe like). Within assays of this type, the ability of a test sample toinhibit the binding of labeled ZS33LP to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

As used herein, the term “antibodies” includes polyclonal antibodies,monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)₂and Fab fragments, single chain antibodies, and the like, includinggenetically engineered antibodies. Non-human antibodies may be humanizedby grafting non-human CDRs onto human framework and constant regions, orby incorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. One skilled in the art can generatehumanized antibodies with specific and different constant domains (i.e.,different Ig subclasses) to facilitate or inhibit various immunefunctions associated with particular antibody constant domains.Antibodies are defined to be specifically binding if they bind to aZS33LP polypeptide or protein with an affinity at least 10-fold greaterthan the binding affinity to control (non-ZS33LP) polypeptide orprotein. The affinity of a monoclonal antibody can be readily determinedby one of ordinary skill in the art (see, for example, Scatchard, Ann.NY Acad. Sci. 51: 660-672, 1949).

As used herein the term “binding partner” refers to any peptide that canbind specifically to the ZS33LP peptides of the present invention. Suchpeptides include, for example, receptors, receptor-like complementarymolecules, and antibodies.

Some members of the gut peptide hormones have been shown to bind to Gprotein coupled receptors (GPCR). See Feighner, S. D. et al., Science284: 2184-2188, 1999; and Kojima, M. et al., Nature 402: 656-660, 1999.Thus, the ZS33LP polypeptides of the present invention may bind a GPCR.Binding of the ZS33LP, or variant thereof, to the GPCR causes the Gprotein to release its bound GDP and to bind GTP, thus, activating the Gprotein. In general, activated G proteins then bind to an affectorenzyme which catalyzes the formation of a second messenger. In the caseof a ZS33LP binding to a GPCR the result of such second messenger can bemeasured by biological events such as, for example, gastriccontractility, modulation of nutrient uptake, modulation of growthhormones, modulation of the secretion of digestive enzymes and hormones,and/or modulation of secretion of enzymes and/or hormones in thepancreas, as well as by other assays discussed herein.

Cells expressing functional GPCRs are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in the target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycalorimetric assay based on the metabolic breakdown of Alamar Blue™(AccuMed, Chicago, Ill.) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63, 1983).

Another assay of interest measures or detects changes in proliferation,differentiation, development and/or electrical coupling of muscle cellsor myocytes. Additionally, the effects of ZS33LP polypeptides oncell-cell interactions of fibroblasts, myoblasts, nerve cells, whiteblood cells, immune cells, and tumor cells would be of interest tomeasure. Yet other assays examine changes in contractility, andsecretion of hormones and enzymes. Alternative assays are also listedherein.

Additional assays provided by the present invention include the use ofhybrid receptor polypeptides. These hybrid polypeptides fall into twogeneral classes. Within the first class, the intracellular domain of aGPCR is joined to the ligand-binding domain of a second receptor. It ispreferred that the second receptor be a G protein-coupled receptor, suchas the motilin receptor, GPR38, or the zsig33 receptor, GHS-R. Thehybrid receptor will further comprise transmembrane domains, which maybe derived from either receptor. A DNA construct encoding the hybridreceptor is then inserted into a host cell. Cells expressing the hybridreceptor are cultured in the presence of motilin, or zsig33,respectively, and assayed for a response. This system provides a meansfor analyzing signal transduction mediated by the GPCR while usingreadily available ligands. This system can also be used to determine ifparticular cell lines are capable of responding to signals transduced bythe GPCR. A second class of hybrid receptor polypeptides comprise theextracellular (ligand-binding) domain of a GPCR with a cytoplasmicdomain of a second receptor, preferably a G protein-coupled receptor,and transmembrane domains. The transmembrane domains may be derived fromeither receptor. Hybrid receptors of this second class are expressed incells known to be capable of responding to signals transduced by thesecond receptor. Together, these two classes of hybrid receptors enablethe use of a broad spectrum of cell types within receptor-based assaysystems.

Another assay uses cell lines expressing G_(α16) and the calciumsensitive photoprotein, aequorin, in a screening system for agonistactivity. This system (described by Stables, J. et al., Anal. Biochem.252:115-126, 1997) uses the G_(α16) protein to couple with any Gprotein-linked receptor. Binding the receptor results in an increase inintracellular calcium concentrations. The cells are pre-incubated incoelenterazine and the intracellular calcium reacts with aequorin (whichhas also been transfected into the cells) and coelenterazine resultingin a luminescent response. Cell lines from pituitary, hypothalamus, andpancreas would be useful for GHS-R in this assay.

The ZS33LP peptides of the present invention may bind the growth hormonesecretagogue receptor. The release of growth hormone stimulates growthin many tissues and has effects on metabolic processes such asstimulating protein synthesis and free fatty acid mobilization as wellas stimulating metabolism from a variety of energy sources fromcarbohydrates to fatty acids. Deficiency of growth hormone can result inmedical disorders such as dwarfism. Growth hormone secretagogues are aclass of small peptides which stimulate the release of growth hormonefrom pituitary cells by a mechanism of action other than that of GHRH,i.e., by binding a different receptor (GHS-R) in the pituitary andhypothalamus. Thus, the binding of this receptor can play a role inregulating growth hormone secretion in extraneuroendocrine activities,such as, for example, sleep and food intake. Therefore, the secretion ofgrowth hormone can be regulated by the formation of a peptide-receptorcomplex between ZS33LP and GHS-R.

The binding of Zs33LP polypeptides to the GSH-R can be measured using avariety of assays that measure, for example, cell-cell interactions;ligand-receptor binding, and other biological functions associated withgut-hormone family members. Of particular interest is a change ingastrointestinal contractility, modulation of growth hormones, weightmaintenance, and glucose absorption. Assays measuring ligand binding andgastrointestinal contractility are known in the art, and furtherdescribed in the examples, herein. Additional assays for measuringgrowth hormone secretion, receptor binding, and body weight aredescribed in Hansen, B. S. ibid.

Proteins, including peptides resulting from alternative splicing, of thepresent invention are useful for modulation of gastrointestinalcontractility, modulation of nutrient uptake, modulation of growthhormones, modulation of the secretion of digestive enzymes and hormones,and/or modulation of secretion of enzymes and/or hormones in thepancreas, and gastric reflux either working in isolation, or inconjunction with other molecules (growth factors, cytokines, etc.) intissues such as stomach, duodenum, jejunum, kidney, small intestine,skeletal muscle, lung, pituitary, hypothalamus, hippocampus, and centralnervous system, in general. Alternative splicing of ZS33LP mRNA may becell-type specific and confer activity to specific tissues.

The effects of ZS33LPs, their antagonists and agonists, on tissuecontractility can be measured in vitro using a tensiometer with orwithout electrical field stimulation. Such assays are known in the artand can be applied to tissue samples, such as gastrointestinal and othercontractile tissue samples, and can be used to determine whetherZS33LPs, their agonists or antagonists, enhance or depresscontractility. Molecules of the present invention are hence useful fortreating dysfunction associated with contractile tissues or can be usedto suppress or enhance contractility in vivo. As such, molecules of thepresent invention have utility in treating gastrointestinal and growthrelated diseases.

The effect of the ZS33LPs, antagonists and agonists of the presentinvention on contractility of tissues including gastrointestinal tissuescan be measured in a tensiometer that measures contractility andrelaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767, 1990;Rhee et al., Neurotox. 16: 179, 1995; Anderson, M. B., Endocrinol.114:364-368, 1984; and Downing, S. J. and Sherwood, O. D, Endocrinol.116:1206-1214, 1985. For example, measuring vasodilatation of aorticrings is well known in the art. Briefly, aortic rings are taken from 4month old Sprague Dawley rats and placed in a buffer solution, such asmodified Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO₄.7H₂O,1.2 mM KH₂PO₄, 2.5 mM CaCl₂.2H₂O, 24.8 mM NaHCO₃ and 10 mM glucose). Oneof skill in the art would recognize that this method can be used withother animals, such as rabbits, other rat strains, Guinea pigs, and thelike. The rings are then attached to an isometric force transducer(Radnoti Inc., Monrovia, Calif.) and the data recorded with a Ponemahphysiology platform (Gould Instrument systems, Inc., Valley View, Ohio)and placed in an oxygenated (95% O₂, 5% CO₂) tissue bath containing thebuffer solution. The tissues are adjusted to 1 gram resting tension andallowed to stabilize for about one hour before testing. The integrity ofthe rings can be tested with norepinepherin (Sigma Co., St. Louis, Mo.)and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). Afterintegrity is checked, the rings are washed three times with fresh bufferand allowed to rest for about one hour. To test a sample forvasodilatation, or relaxation of the aortic ring tissue, the rings arecontracted to two grams tension and allowed to stabilize for fifteenminutes. A ZS33LP sample is then added to 1, 2 or 3 of the 4 baths,without flushing, and tension on the rings recorded and compared to thecontrol rings containing buffer only. Enhancement or relaxation ofcontractility by ZS33LPs, their agonists and antagonists is directlymeasured by this method, and it can be applied to other contractiletissues such as gastrointestinal tissues.

Potential uses of growth hormone are extensive and include treatment ofdiseases and conditions associated with bone formation (such as, forexample, treatment of osteoporosis, acceleration of bone formation andrepair, stimulating osteoblasts, bone remodeling and cartilage growth,and skeletal dysplasia); immunity (such as, for example, stimulating theimmune system, treating immunosuppressed patients); obesity, andmetabolic disorders (such as, for example, preventing catabolic sideeffects of glucocorticoids, treatment of obesity and growth retardationrelated to obesity, attenuation of protein catabolic responses aftersurgery, reducing cachexia and protein loss due to chronic illness suchas cancer or AIDS); dwarfism (such as, for example, treating growthretardation and physiological short stature including growth hormonedeficiency and chromic illness, and intrauterine growth retardation);wound healing (such as, for example, accelerating wound repair,accelerating recovery of burn patients and treating patients withdelayed wound healing); reproduction (such as, for example, as anadjuvant treatment for ovulation induction); as well as conditionsassociated with stress; conditions associated with kidney and lungdysfunction; conditions associated with aging and the elderly,including, muscle strength, bone fragility and skin thickness; andneuroendocrine activities such as sleep. Thus, growth hormonesecretagogues are useful to treat conditions associated with thesedisorders. Assays measuring the release of growth hormone are known inthe art.

An association between gastrointestinal function and brain function hasbeen observed for other hormones in this class. As an example, secretininfusion in autistic children resulted in amelioration of thegastrointestinal symptoms as well as a dramatic improvement in behavior(improved eye contact, alertness and expansion of expressive language).See Hovrath, K. et al., J. Assoc. Acad. Minor Phys 9(1):9-15, 1998.Similarly, a study of the upper gastrointestinal tract in autisticchildren with gastrointestinal symptoms showed that many had refluxesophagitis, chronic gastritis, and chronic duodenitis, as well as anelevated number of Paneth's cells in the duodenal crypts compared tonon-autistic children. See Horvath, K. et al., J. Pediatr.135(5):559-563, 1999. The administration of secretin to these autisticchildren resulted in increased pancreatico-biliary fluid output andhigher fluid output. Gastrointestinal disorders, especially refluxesophagitis and disaccharide malabsoprtion may contribute to thebehavioral problems of the non-verbal autistic patients. The observedincrease in pancreatico-biliary secretion after secretin infusionsuggests an upregulation of secretin receptors. As a member of thegut-hormone family of proteins, ZS33LP, by binding to its receptor, mayhave effects on neural development and/or utilization.

Molecules of the present invention can be used to identify and isolateother isoforms of GHS-R, or other G protein-coupled receptors,cell-surface binding proteins, or members of complement/anti-complementpairs involved in gut-hormone interactions. For example, ZS33LP can beimmobilized on a column and membrane preparations run over the column(Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,Academic Press, San Diego, Calif., 1992, pp. 195-202). Proteins andpeptides can also be radiolabeled (Methods in Enzymol., vol. 182, “Guideto Protein Purification”, M. Deutscher, ed., Acad. Press, San Diego,1990, 721-37) or photoaffinity labeled (Brunner et al., Ann. Rev.Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.33:1167-80, 1984) and specific cell-surface proteins can be identified.

ZS33LPs, variants, and fragments thereof, may be useful as replacementtherapy for disorders associated with cell-cell interactions, includingdisorders related to, for example, stimulation of gastrointestinalcontractility, modulation of nutrient uptake, modulation of growthhormones, modulation of the secretion of digestive enzymes and hormones,and modulation of secretion of enzymes and/or hormones in the pancreas.

A less widely appreciated determinant of tissue morphogenesis is theprocess of cell rearrangement: Both cell motility and cell-cell adhesionare likely to play central roles in morphogenetic cell rearrangements.Cells need to be able to rapidly break and probably simultaneouslyremake contacts with neighboring cells. See Gumbiner, B. M., Cell69:385-387, 1992. As a secreted protein in stomach, pituitary,hypothalamus, hippocampus, lung, kidney, duodenum, jejunum, smallintestine, skeletal muscle, and pancreas, ZS33LPs may play a role inintercellular rearrangement in these and other tissues.

ZL33LP binding proteins, such as an anti-ZS33LP antibody, or a GPCR, mayalso be used within diagnostic systems for the detection of circulatinglevels of ZS33LP. Elevated or depressed levels of ligand or receptorpolypeptides may be indicative of pathological conditions, includingcancer.

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982, which is incorporated herein by reference). Ofparticular interest are generating antibodies to hydrophilic antigenicsites which include, for example, residues 6-22 of SEQ ID NO:4, residues14-22 of SEQ ID NO:7, and residues 8 to 17 of SEQ ID NO:10. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from a variety of warm-blooded animals such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. Theimmunogenicity of a ZS33LP polypeptide may be increased through the useof an adjuvant such as alum (aluminum hydroxide) or Freund's complete orincomplete adjuvant. Polypeptides useful for immunization also includefusion polypeptides, such as fusions of a ZS33LP polypeptide or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Alternative techniques for generating or selecting antibodies include invitro exposure of lymphocytes to ZS33LP polypeptides, and selection ofantibody display libraries in phage or similar vectors (e.g., throughthe use of immobilized or labeled ZS33LP polypeptides). Human antibodiescan be produced in transgenic, non-human animals that have beenengineered to contain human immunoglobulin genes as disclosed in WIPOPublication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

A variety of assays known to those skilled in the art can be utilized todetect antibodies that specifically bind to ZS33LP polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, Westernblot assays, inhibition or competition assays, and sandwich assays.

Antibodies to ZS33LP may be used for affinity purification of theprotein, within diagnostic assays for determining circulating levels ofthe protein; for detecting or quantitating soluble ZS33LP polypeptide asa marker of underlying pathology or disease; for neutralizing theeffects of ZS323LP, for immunolocalization within whole animals ortissue sections, including immunodiagnostic applications; forimmunohistochemistry; and as antagonists to block protein activity invitro and in vivo. Antibodies to ZS33LP may also be used for taggingcells that express ZS33LP; for affinity purification of ZS33LPpolypeptides and proteins; in analytical methods employing FACS; forscreening expression libraries; and for generating anti-idiotypicantibodies. Antibodies can be linked to other compounds, includingtherapeutic and diagnostic agents, using known methods to provide fortargeting of those compounds to cells expressing receptors for ZS33LP.For certain applications, including in vitro and in vivo diagnosticuses, it is advantageous to employ labeled antibodies. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies of the present invention may also be directlyor indirectly conjugated to drugs, toxins, radionuclides and the like,and these conjugates used for in vivo diagnostic or therapeuticapplications (e.g., inhibition of cell proliferation). See, in general,Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

Polypeptides and proteins of the present invention can be used toidentify and isolate receptors. ZS33LP receptors may be involved ingrowth regulation in the liver, blood vessel formation, and otherdevelopmental processes. For example, ZS33LP proteins and polypeptidescan be immobilized on a column, and membrane preparations run over thecolumn (as generally disclosed in Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp. 195-202). Proteins and polypeptides can also be radiolabeled(Methods Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) orphotoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514,1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and usedto tag specific cell-surface proteins. In a similar manner, radiolabeledZS33LP proteins and polypeptides can be used to clone the cognatereceptor in binding assays using cells transfected with an expressioncDNA library.

The peptides, variants, nucleic acid and/or antibodies of the presentinvention may be used in treatment of disorders associated withgastrointestinal contractility, secretion of digestive enzymes, hormonesand acids, secretion of hormones in the pancreas and/or brain,gastrointestinal motility, recruitment of digestive enzymes;inflammation, particularly as it affects the gastrointestinal system;reflux disease and regulation of nutrient absorption. Specificconditions that will benefit from treatment with molecules of thepresent invention include, but are not limited to, diabeticgastroparesis, post-surgical gastroparesis, vagotomy, chronic idiopathicintestinal pseudo-obstruction and gastroesophageal reflux disease.Additional uses include, gastric emptying for radiological studies,stimulating gallbladder contraction and antrectomy.

The motor and neurological affects of molecules of the present inventionmake it useful for treatment of obesity and other metabolic disorderswhere neurological feedback modulates nutritional absorption. Themolecules of the present invention are useful for regulating satiety,glucose absorption and metabolism, and neuropathy-associatedgastrointestinal disorders.

Peptides of the present invention may be useful for evaluating functionsof the hypothalamus-pituitary-adrenal axis by challenging thegastrointestinal system with zsig33-beta and zsig33-gamma peptides,including variants, and measuring gastric motility and contractility,modulation of nutrient uptake, modulation of the secretion of digestiveenzymes and hormones, or modulation of secretion of enzymes and/orhormones in the pancreas.

Additionally, molecules of zsig33-beta and zsig33-gamma peptides may beused to detect or modulate the growth and/or differentiation of tumorcells which are expressing a receptor which binds to zsig33-beta andzsig33-gamma peptides. zsig33-beta and zsig33-gamma peptides can belabeled with radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. These labeledpolypeptides can be applied in vitro or in vivo and are especiallyuseful to identify receptors for zsig33-beta, zsig33-gamma,zsig33-delta, and zsig33-epsilon located on tumors in such tissues as,for example, stomach, brain, pancreas, kidney, duodenum, jejunum, andlung.

Molecules of the present invention are also useful as additives toanti-hypoglycemic preparations containing glucose and as adsorptionenhancers for oral drugs which require fast nutrient action.Additionally, molecules of the present invention can be used tostimulate glucose-induced insulin release.

A common side effect experience by livestock raised in feedlot settingsis failure to feed shortly after transport and/or post illness or changein environments. Often feed additives are used to stimulate gastricmotility to encourage the animal to feed. Thus, molecules of the presentinvention can be used alone or in conjunction with existing therapies tostimulate hunger and feeding. Such results can be seen both in healthyand challenged animals.

Inhibitors of ZS33LP activity (ZS33LP antagonists) include anti-ZS33LPantibodies and soluble ZS33LP receptors, as well as other peptidic andnon-peptidic agents (including ribozymes). Such antagonists can be usedto block the effects of ZS33LP on cells or tissues. Of particularinterest is the use of antagonists of ZS33LP activity in conditions suchas Inflammatory or Irritable Bowel syndromes. Inhibitors can also beused in combination with other therapeutic agents.

In addition to antibodies, ZS33LP inhibitors include small moleculeinhibitors and inactive receptor-binding fragments of ZS33LPpolypeptides. Inhibitors are formulated for pharmaceutical use asgenerally disclosed above, taking into account the precise chemical andphysical nature of the inhibitor and the condition to be treated. Therelevant determinations are within the level of ordinary skill in theformulation art.

Alternatively, ZS33LP may activate the immune system which would beimportant in boosting immunity to infectious diseases, treatingimmunocompromised patients, such as HIV+ patients, or in improvingvaccines. In particular, ZS33LP stimulation or expansion ofhematopoietic cells, or their progenitors, would provide therapeuticvalue in treatment of bacterial or viral infection, and as ananti-neoplastic factor. ZS33LP stimulation of the immune responseagainst viral and non-viral pathogenic agents (including bacteria,protozoa, and fungi) would provide therapeutic value in treatment ofsuch infections by inhibiting the growth of such infections agents.Determining directly or indirectly the levels of a pathogen or antigen,such as a tumor cell, present in the body can be achieved by a number ofmethods known in the art and described herein.

The present invention includes a method of stimulating an immuneresponse in a mammal exposed to an antigen or pathogen comprising thesteps of: (1) determining directly or indirectly the level of antigen orpathogen present in said mammal; (2) administering a compositioncomprising a ZS33LP polypeptide in an acceptable pharmaceutical vehicle;(3) determining directly or indirectly the level of antigen or pathogenin said mammal; and (4) comparing the level of the antigen or pathogenin step 1 to the antigen or pathogen level in step 3, wherein a changein the level is indicative of stimulating an immune response. In anotherembodiment the ZS33LP composition is re-administered. In otherembodiments, the antigen is a B cell tumor; a virus; a parasite or abacterium.

In another aspect, the present invention provides a method ofstimulating an immune response in a mammal exposed to an antigen orpathogen comprising: (1) determining a level of an antigen- orpathogen-specific antibody; (2) administering a composition comprisingZS33LP polypeptide in an acceptable pharmaceutical vehicle; (3)determining a post administration level of antigen- or pathogen-specificantibody; (4) comparing the level of antibody in step (1) to the levelof antibody in step (3), wherein an increase in antibody level isindicative of stimulating an immune response.

Polynucleotides encoding ZS33LP polypeptides are useful within genetherapy applications where it is desired to increase or inhibit ZS33LPactivity. If a mammal has a mutated or absent ZS33LP gene, a ZS33LP genecan be introduced into the cells of the mammal. In one embodiment, agene encoding a ZS33LP polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas, but not limited to, herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes simplex virus 1(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991);an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest. 90:626-630, 1992; and adefective adeno-associated virus vector (Samulski et al., J. Virol.61:3096-3101, 1987; Samulski et al., J. Virol. 63:3822-3888, 1989).Within another embodiment, ZS33LP gene can be introduced in a retroviralvector as described, for example, by Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; Doughertyet al., WIPO Publication WO 95/07358; and Kuo et al., Blood 82:845,1993. Alternatively, the vector can be introduced by liposome-mediatedtransfection (“lipofection”). Synthetic cationic lipids can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackeyet al., Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988). The use oflipofection to introduce exogenous genes into specific organs in vivohas certain practical advantages, including molecular targeting ofliposomes to specific cells. Directing transfection to particular celltypes is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Peptidic and non-peptidic molecules can be coupled toliposomes chemically. Within another embodiment, cells are removed fromthe body, a vector is introduced into the cells as a naked DNA plasmid,and the transformed cells are re-implanted into the body as disclosedabove. Antisense methodology can be used to inhibit ZS33LP genetranscription in a patient as generally disclosed above.

ZS33LP polypeptides and anti-Zs33LP antibodies can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention may be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, ZS33LPpolypeptides or anti-ZS33LP antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues, or organs that expressthe anti-complementary molecule.

Suitable detectable molecules can be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles, and the like. Suitable cytotoxic moleculescan be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 oryttrium-90. These can be either directly attached to the polypeptide orantibody, or indirectly attached according to known methods, such asthrough a chelating moiety. Polypeptides or antibodies can also beconjugated to cytotoxic drugs, such as adriamycin. For indirectattachment of a detectable or cytotoxic molecule, the detectable orcytotoxic molecule may be conjugated with a member of acomplementary/anticomplementary pair, where the other member is bound tothe polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusionproteins may be used for targeted cell or tissue inhibition or ablation,such as in cancer therapy. Of particular interest in this regard areconjugates of a ZS33LP polypeptide and a cytotoxin, which can be used totarget the cytotoxin to a tumor or other tissue that is undergoingundesired angiogenesis or neovascularization. Target cells (i.e., thosedisplaying the ZS33LP) bind the ZS33LP-toxin conjugate, which is theninternalized, killing the cell. The effects of receptor-specific cellkilling (target ablation) are revealed by changes in whole animalphysiology or through histological examination. Thus, ligand-dependent,receptor-directed cyotoxicity can be used to enhance understanding ofthe physiological significance of a protein ligand. A preferred suchtoxin is saporin. Mammalian cells have no receptor for saporin, which isnon-toxic when it remains extracellular.

In another embodiment ZS33LP-cytokine fusion proteins orantibody/fragment-cytokine fusion proteins may be used for enhancing invitro cytotoxicity (for instance, that mediated by monoclonal antibodiesagainst tumor targets) and for enhancing in vivo killing of targettissues (for example, blood and bone marrow cancers). See, generally,Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines aretoxic if administered systemically. The described fusion proteins enabletargeting of a cytokine to a desired site of action, such as a cellhaving binding sites for ZS33LP, thereby providing an elevated localconcentration of cytokine. Suitable cytokines for this purpose include,for example, interleukin-2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF). Such fusion proteins may be used to causecytokine-induced killing of tumors and other tissues undergoingangiogenesis or neovascularization.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intra-arterially or intraductally, or may beintroduced locally at the intended site of action.

ZS33LPs of the present invention may be useful for evaluating functionsof the hypothalamus-pituitary-adrenal axis by challenging thegastrointestinal system with ZS33LPs, including variants, and measuringgastric motility and contractility, modulation of nutrient uptake,modulation of growth hormones, modulation of the secretion of digestiveenzymes and hormones, or modulation of secretion of enzymes and/orhormones in the pancreas.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Chemical Synthesis and Purification of Zsig33-Linker,Zsig33-Gamma, and Zsig33-Delta, Peptides

Zsig33-linker peptide, a peptide corresponding to amino acid residue 1(Ala) to amino acid residue 24 (Arg) of SEQ ID NO: 4, is synthesized bysolid phase peptide synthesis using a model 431A Peptide Synthesizer(Applied Biosystems/Perkin Elmer, Foster City, Calif.). Fmoc-Glutamineresin (0.63 mmol/g; Advanced Chemtech, Louisville, Ky.) is used as theinitial support resin. 1 mmol amino acid cartridges (Anaspec, Inc. SanJose, Calif.) are used for synthesis. A mixture of2(1-Hbenzotriazol-y-yl 1,1,3,3-tetrahmethylhyluroniumhexafluorophosphate (HBTU), 1-hydroxybenzotriazol (HOBt), 2 mN,N-Diisolpropylethylamine, N-Methylpyrrolidone, Dichloromethane (allfrom Applied Biosystems/Perkin Elmer) and piperidine (Aldrich ChemicalCo., St. Louis, Mo.), and used for synthesis reagents.

The Peptide Companion software (Peptides International, Louisville, Ky.)is used to predict the aggregation potential and difficulty level forsynthesis for the zsig33-linker peptide. Synthesis is performed usingsingle coupling programs, according to the manufacturer'sspecifications.

The peptide is cleaved from the solid phase following standard TFAcleavage procedure (according to Peptide Cleavage manual, AppliedBiosystems/Perkin Elmer). Purification of the peptide is done by RP-HPLCusing a C18, 10 μm semi-peparative column (Vydac, Hesperial, CA). Elutedfractions from the column are collected and analyzed for correct massand purity by electrospray mass spectrometry. Two pools of the elutedmaterial are collected. Purity is verified by mass spectrometryanalysis.

A zsig33-gamma peptide corresponding to residues 1 to 17 of SEQ IDNO:10, and a zsig33-deltapeptide corresponding to residues 1 to 10 ofSEQ ID NO:14 are synthesized by the same method.

Example 2 Chemical Synthesis and Purification of Zsig33-Beta andZsig33-Epsilon Peptides

A zsig33-beta polypeptide, such as that shown in SEQ ID NOs:4, 5 and 6,is synthesized by solid phase peptide synthesis using the ABI/PE PeptideSynthesizer model 431A (Applied Biosytems/Perkin Elmer (ABI/PE, FosterCity, Calif.).

Fmoc-Amide resin is used for synthesis of the active zsig33-betapeptide-amide and Fmoc-Asparagine resin are used for non-amidatedzsig33-beta peptides. The Fmoc-Amide resin (0.68 mmol/g) and theFmoc-Asparagine resin (0.75 mmol/g) are purchased from ABI/PE. The aminoacids can be purchased from AnaSpec, Inc., San Jose, Calif. inpre-weighed, 1 mmol cartridges. All the reagents except piperidine arepurchased from ABI/PE. The piperidine is purchased from Aldrich, St.Louis Mo. Synthesis procedure is taken from the ABI Model 431A manual.Double coupling cycles are used during the high aggregation portion ofthe sequence, as predicted by Peptide Companion software (PeptidesInternational, Louisville, Ky.).

The peptides are cleaved from the solid phase following the standard TFAcleavage procedure as outlined in the Peptide Cleavage protocol manualpublished by ABI/PE. Purification of the peptides is by RP-HPLC using aC18, 10 mm preparative column. Eluted fractions from the column arecollected and analyzed for correct mass and purity by electrospray massspectrometry. The analysis results should indicate that the Zsig33-betapeptide is present and pure in one of the pools from the HPLCpurification step. The pools containing each of the peptides is retainedand lyophilized.

Post lyophilization, the zsig33-beta peptide is analyzed for purityusing analytical HPLC. The analytical HPLC column used is a Vydac 10 cm,5 um column. The analysis should result in 95% purity for Zsig33-betapeptides. These peptide is prepared for use in subsequent biologicalassays.

A zsig33-epsilon peptide corresponding to residues 1 to 13 of SEQ IDNO:17, is synthesized by the same method.

Example 3 Construction of Expression Plasmids

An expression plasmid containing all or part of a polynucleotideencoding a ZS33LP protein is constructed via homologous recombination. Afragment of ZS33LP cDNA is isolated by PCR using the polynucleotidesequence of the zsig33 secretion leader (nucleotides 50 to 118 of SEQ IDNO:1) followed by the polynucleotide sequence of SEQ ID NO: 3, 6, 9, 13,or 16 with flanking regions at the 5′ and 3′ ends corresponding to thevector sequences flanking the ZS33LP insertion point. The primers forPCR each include from 5′ to 3′ end: 40 bp of flanking sequence from thevector and 17 bp corresponding to the amino and carboxyl termini fromthe open reading frame of the ZS33LP.

Ten μl of the 100 μl PCR reaction mixture is run on a 0.8%low-melting-temperature agarose (SeaPlaque GTG®; FMC BioProducts,Rockland, Me.) gel with 1×TBE buffer for analysis. The remaining 90 μlof the reaction mixture is precipitated with the addition of 5 μl 1 MNaCl and 250 μl of absolute ethanol. The plasmid pZMP6, which has beencut with SmaI, is used for recombination with the PCR fragment. PlasmidpZMP6 is a mammalian expression vector containing an expression cassettehaving the cytomegalovirus immediate early promoter, multiplerestriction sites for insertion of coding sequences, a stop codon, and ahuman growth hormone terminator; an E. coli origin of replication; amammalian selectable marker expression unit comprising an SV40 promoter,enhancer and origin of replication, a DHFR gene, and the SV40terminator; and URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae. It is constructed from pZP9 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 98668) with the yeastgenetic elements taken from pRS316 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Accession No. 77145), an internal ribosome entry site(IRES) element from poliovirus, and the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain.

One hundred microliters of competent yeast (S. cerevisiae) cells areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply (BioRad Laboratories,Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated intwo 300-μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura⁺ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid-ished glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA is precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet is resuspended in10 μl H₂O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression constructs forzsig33-linker, zsig33-beta, zsig33-gamma, zsig33-delta, andzsig33-epsilon are identified by restriction digest to verify thepresence of the correct insert and to confirm that the various DNAsequences have been joined correctly to one another. The inserts ofpositive clones are subjected to sequence analysis. Larger scale plasmidDNA is isolated using a commercially available kit (QIAGEN Plasmid MaxiKit, Qiagen, Valencia, Calif.) according to manufacturer's instructions.The correct constructs are designated pZMP6/zsig33-linker, zsig33-beta,zsig-33 gamma, zsig33-delta, and zsig33-epsilon.

Example 4 Expression in Chinese Hamster Ovary Cells

CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10-cm tissue culture dishes and allowed to grow toapproximately 50% to 70% confluency overnight at 37° C., 5% CO₂, inHam's F12/FBS media (Ham's F12 medium (Life Technologies), 5% fetalbovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences,Lenexa, Kans.), 1% sodium pyruvate (Life Technologies)). The cells arethen transfected with the plasmids from Example 3 by liposome-mediatedtransfection using a 3:1 (w/w) liposome formulation of the polycationiclipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filtered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zsig33-linker, zsig33-beta, and zsig-33 gamma/pZMP6 is diluted into15-ml tubes to a total final volume of 640 μl with SF media. 35 μl ofLipofectamine™ is mixed with 605 μl of SF medium. The resulting mixtureis added to the DNA mixture and allowed to incubate approximately 30minutes at room temperature. Five ml of SF media is added to theDNA:Lipofectamine™ mixture. The cells are rinsed once with 5 ml of SFmedia, aspirated, and the DNA:Lipofectamine™ mixture is added. The cellsare incubated at 37° C. for five hours, then 6.4 ml of Ham's F12/10%FBS, 1% PSN media is added to each plate. The plates are incubated at37° C. overnight, and the DNA:Lipofectamine™ mixture is replaced withfresh 5% FBS/Ham's media the next day. On day 3 post-transfection, thecells are split into T-175 flasks in growth medium. On day 7post-transfection, the cells are stained with FITC-anti-CD8 monoclonalantibody (Pharmingen, San Diego, Calif.) followed byanti-FITC-conjugated magnetic beads (Miltenyi Biotec). The CD8-positivecells are separated using commercially available columns (mini-MACScolumns; Miltenyi Biotec) according to the manufacturer's directions andput into DMEM/Ham's F12/5% FBS without nucleosides but with 50 nMmethotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells perwell in 96-well dishes in selection medium and allowed to grow out forapproximately two weeks. The wells are checked for evaporation of mediumand brought back to 200 μl per well as necessary during this process.When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is then ished three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per ish. The filteris developed with chemiluminescence reagents (ECL™ direct labelling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham Corp.) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to 6-well dishes in selection medium forscaleup and analysis by Western blot.

Example 5 Expression in Baby Hamster Kidney Cells

ZS33LPs are produced in BHK cells transfected with the plasmids preparedin Example 3. BHK 570 cells (ATCC CRL-10314) are plated in 10-cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluenceovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose; Life Technologies), 5% fetal bovine serum (Hyclone, Logan,Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodiumpyruvate (Life Technologies). The cells are then transfected withpZMP6/zsig33-linker, zsig33-beta, and zsig33-gamma by liposome-mediatedtransfection (using Lipofectamine™; Life Technologies), in serum free(SF) media (DMEM supplemented with 10 mg/ml transferrin, 5 mg/mlinsulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). Theplasmid is diluted into 15-ml tubes to a total final volume of 640 μlwith SF media. 35 μl of the lipid mixture is mixed with 605 μl of SFmedium, and the resulting mixture is allowed to incubate approximately30 minutes at room temperature. Five milliliters of SF media is thenadded to the DNA:lipid mixture. The cells are rinsed once with 5 ml ofSF media, aspirated, and the DNA:lipid mixture is added. The cells areincubated at 37° C. for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSNmedia is added to each plate. The plates are incubated at 37° C.overnight, and the DNA:lipid mixture is replaced with fresh 5% FBS/DMEMmedia the next day. On day 5 post-transfection, the cells are split intoT-162 flasks in selection medium (DMEM+5% FBS, 1% L-Gln, 1% NaPyr, 1 μMmethotrexate). Approximately 10 days post-transfection, two 150-mmculture dishes of methotrexate-resistant colonies from each transfectionare trypsinized, and the cells are pooled and plated into a T-162 flaskand transferred to large-scale culture.

Example 6 Protein Purification

Purification conditions for ZS33LPs with N- and C-terminal EE tags:

E. coli, Pichia, CHO and BHK cells are transfected with expressionplasmids constructed in Example 3 and operably linked to apolynucleotide encoding a Glu-Glu tag (SEQ ID NO:28). ZS33LPs areexpressed in the conditioned media of the E. coli, Pichia methanolica,and or chinese hamster ovary (CHO) cells. For ZS33LPs expressed in E.coli and Pichia, the media is not concentrated prior to purification.Unless otherwise noted, all operations are carried out at 4° C. A totalof 25 liters of conditioned medium from BHK cells is sequentiallysterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, Mass.)OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, Mich.) Supercap50. The material is then concentrated to about 1.3 liters using aMillipore ProFlux A30 tangential flow concentrator fitted with a 3000kDa cutoff Amicon (Bedford, Mass.) S1OY3 membrane. The concentratedmaterial is again sterile-filtered with the Gelman filter, as describedabove. A mixture of protease inhibitors is added to the concentratedconditioned medium to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). A 50.0 ml sample of anti-EE Sepharose, preparedas described below, is added and the mixture gently agitated on aWheaton (Millville, N.J.) roller culture apparatus for 18.0 h at 4° C.

The mixture is then poured into a 5.0×20.0 cm Econo-Column (Bio-Rad,Laboratories, Hercules, Calif.), and the gel is ished with 30 columnvolumes of phosphate buffered saline (PBS). The unretained flow-throughfraction is discarded. Once the absorbance of the effluent at 280 nM isless than 0.05, flow through the column is reduced to zero, and theanti-EE Sepharose gel is ished with 2.0 column volumes of PBS containing0.2 mg/ml of EE peptide (AnaSpec, San Jose, Calif.). The peptide that isused has the sequence GluTyrMetProValAsp. After 1.0 h at 4° C., flow isresumed and the eluted protein collected. This fraction is referred toas the peptide elution. The anti-EE Sepharose gel is then ished with 2.0column volumes of 0.1 M glycine, pH 2.5, and the glycine ish iscollected separately. The pH of the glycine-eluted fraction is adjustedto 7.0 by the addition of a small volume of 10×PBS and stored at 4° C.for future analysis, if needed.

The peptide elution is concentrated to 5.0 ml using a 15,000 molecularweight cutoff membrane concentrator (Millipore, Bedford, Mass.),according to the manufacturer's instructions. The concentrated peptideelution is then separated from free peptide by chromatography on a1.5×50 cm Sephadex G-50 (Pharmacia, Piscataway, N.J.) columnequilibrated in PBS at a flow rate of 1.0 mL/min using a BioCad SprintHPLC (PerSeptive BioSystems, Framingham, Mass.). Two-ml fractions arecollected and the absorbance at 280 nM monitored. The first peak ofmaterial absorbing at 280 nM and eluting near the void volume of thecolumn is collected. This fraction is pure ZS33LP NEE or ZS33LP CEE. Thepure material is concentrated as described above, analyzed by SDS-PAGEand Western blotting with anti-EE antibodies, aliquoted, and stored at−80° C. according to standard procedures.

Preparation of Anti-EE Sepharose:

A 100 ml bed volume of protein G-Sepharose (Pharmacia, Piscataway, N.J.)is ished 3 times with 100 ml of PBS containing 0.02% sodium azide usinga 500 ml Nalgene 0.45 micron filter unit. The gel is ished with 6.0volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.).and an equal volume of EE antibody solution containing 900 mg ofantibody is added. After an overnight incubation at 4° C., unboundantibody is removed by ishing the resin with 5 volumes of 200 mM TEA asdescribed above. The resin is resuspended in 2 volumes of TEA,transferred to a suitable container, and dimethylpimilimidate-2HCl(Pierce, Rockford, Ill.), dissolved in TEA, is added to a finalconcentration of 36 mg/ml of gel. The gel is rocked at room temperaturefor 45 min and the liquid is removed using the filter unit as describedabove. Nonspecific sites on the gel are then blocked by incubating for10 min at room temperature with 5 volumes of 20 mM ethanolamine in 200mM TEA. The gel is then ished with 5 volumes of PBS containing 0.02%sodium azide and stored in this solution at 4° C.

Purification of Untagged zsig33:

E. coli, Pichia, CHO and BHK cells are transfected with expressionvectors containing the DNA sequence of SEQ ID NO:1, or a portionthereof. The procedure described below is used for protein expressed inconditioned medium of E. coli, Pichia methanolica, and Chinese hamsterovary (CHO) and baby hamster kidney (BHK) cells. For ZS33LP expressed inE. coli and Pichia, however, the medium is not be concentrated prior topurification. Unless otherwise noted, all operations are carried out at4° C. A total of 25 liters of conditioned medium from BHK cells issequentially sterile filtered through a 4 inch, 0.2 mM Millipore(Bedford, Mass.) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor,Mich.) Supercap 50. The material is then be concentrated to about 1.3liters using a Millipore ProFlux A30 tangential flow concentrator fittedwith a 3000 kDa cutoff Amicon (Bedford, Mass.) S1OY3 membrane. Theconcentrated material is again sterile-filtered with the Gelman filteras described above. A mixture of protease inhibitors is added to theconcentrated conditioned medium to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim).

Example 7 Construction of BaF3 Cells Expressing a GPCR

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), ismaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, pZP-5N/GHS-R cDNA (Example 5) isprepared and purified using a Qiagen Maxi Prep kit (Qiagen) as permanufacturer's instructions. BaF3 cells for electroporation are ishedonce in RPMI media and then resuspended in RPMI media at a cell densityof 10⁷ cells/ml. One ml of resuspended BaF3 cells is mixed with 30 μg ofa plasmid containing a GPCR (such as for example, GP38, or GHS-R) andtransferred to separate disposable electroporation chambers (GIBCO BRL).Following a 15 minute incubation at room temperature the cells are giventwo serial shocks (800 1Fad/300 V.; 1180 1Fad/300 V.) delivered by anelectroporation apparatus (CELL-PORATOR™; GIBCO BRL). After a 5 minuterecovery time, the electroporated cells are transferred to 50 ml ofcomplete media and placed in an incubator for 15-24 hours (37° C., 5%CO₂). The cells are then spun down and resuspended in 50 ml of completemedia containing Geneticin™ (Gibco) selection (500 μg/ml G418) in aT-162 flask to isolate the G418-resistant pool. Pools of the transfectedBaF3 cells, are assayed for ZS33LP binding capability as describedbelow.

Example 8 Screening for GPCR Activity Using BaF3/GPCR Cells Using anAlamar Blue Proliferation Assay

BaF3/GPCR cells are spun down and ished in mIL-3 free media 3 times toensure the removal of the mIL-3. Cells are then counted in ahemacytometer, and plated in a 96-well format at 5000 cells per well ina volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/GPCR cells is assessed using media containingsynthesized ZS33LPs which have been diluted with mIL-3 free media to50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations.100 μl of the diluted synthesized ZS33LP is added to the BaF3/GPCRcells. The total assay volume is 200 μl. Negative controls are run inparallel using mIL-3 free media only, without the addition of ZS33LP.The assay plates are incubated at 37° C., 5% CO₂ for 3 days at whichtime Alamar Blue (Accumed, Chicago, Ill.) is added at 20 μl/well. AlamarBlue gives a fluourometric readout based on number of live cells, and isthus a direct measurement of cell proliferation in comparison to anegative control. Plates are again incubated at 37° C., 5% CO₂ for 24hours. Plates are read on the Fmax™ plate reader (Molecular DevicesSunnyvale, Calif.) using the SoftMax™ Pro program, at wavelengths 544(Excitation) and 590 (Emmission).

A positive result is measured as approximately 4-fold over backgroundwhen the BaF3 wild type cells do not proliferate at the sameconcentration. Additional variants, produced synthetically, orrecombinantly, are also screened in this manner. Similarly, antibodiesto ZS33LP may be tested in this manner for inhibition/antagonism ofZS33LPs.

Example 9 ZS33LP Anti-Peptide Antibodies

Polyclonal anti-peptide antibodies are prepared by immunizing two femaleNew Zealand white rabbits with the ZS33LPs (SEQ ID NOs: 4, 7, 10, 14,and 17). The peptides are synthesized as in Examples 1 and 2. Thepeptides are then conjugated to the carrier protein maleimide-activatedkeyhole limpet hemocyanin (KLH) through cysteine residues (Pierce,Rockford, Ill.). The rabbits are each given an initial intraperitoneal(IP) injection of 200 mg of conjugated peptide in Complete Freund'sAdjuvant (Pierce, Rockford, Ill.) followed by booster IP injections of100 mg conjugated peptide in Incomplete Freund's Adjuvant every threeweeks. Seven to ten days after the administration of the third boosterinjection, the animals are bled and the serum is collected. The rabbitsare then boosted and bled every three weeks.

The ZS33LP-specific antibodies are affinity purified from the rabbitserum using an CNBr-SEPHAROSE 4B peptide column (Pharmacia LKB) that isprepared using 10 mg of the ZS33LP peptide per gram CNBr-SEPHAROSE,followed by dialysis in PBS overnight. Peptide specific-ZS33LPantibodies are characterized by an ELISA titer check using 1 mg/ml ofthe appropriate peptide as an antibody target.

Example 10 Binding Studies of ZS33LP In Situ

Ten week old Balb C male mice are anesthetized via intramuscularinjection and tested for binding of ZS33LP peptides in vivo.

ZS33LPs are tested (i.e., SEQ ID NOs: 4, 7, 10, 14, and 17). A singleglycine is used as a negative control. Additionally, a “scrambled”negative control is also tested. The peptides and controls are coupledto fluorescein isothiocyantate (FITC, Molecular Probes, Eugene, Oreg.)in the following manner: The peptides, glycine control and FITC aredissolved in 0.1 M sodium bicarbonate at pH 9.0 to a concentration of2.0 mg/ml for the peptides and glycine control and 5 mg/ml for FITC,avoiding exposure of the FITC to strong light. The FITC/sodiumbicarbonate solution is added to the peptides at a ratio of 1 mg FITC to1 mg peptide or glycine control, and allowed to react in the dark atambient room temperature for 1 hour. The FITC-conjugated peptides andglycine control are dialyzed in a 1 K dialysis membrane and 0.1 M sodiumbicarbonate buffer at 4° C. The buffer is changed daily and unbound FITCin the post-dialyzed buffer is measured by HPLC. After six days, thebuffer is changed to phosphate buffered saline (PBS) and dialyzed fortwo days followed by another change in PBS and dialyzed for another 2days. Peptide- or glycine-bound FITC is determined by measuring theabsorbance of the dialyzed FITC-bound material at 498 nm and dividing bythe extinction coefficient of fluorescein, 0.083 μM. The molar ratio offluorescein to peptide (mole FITC/mole peptide) is then determined.

The labeled peptides are administered via tail vein injections such thateach mouse received 0.5 ml (0.5 mg) of labeled peptide which is allowedto circulate in the mice for 15 minutes following injection.

While under anesthesia the right atrium of each mouse is snipped toallow an exit path and 20 ml of PBS is injected into left ventricle andused to flush the circulatory system. The mice are then perfused withapproximately 10 ml of formalin in neutral buffer (10% Neutral BufferedFormalin (NBF), Surgipath, Richmond, Ill.).

Tissues are harvested by dissection, and fixed overnight in 10% NBFbefore processing for histological evaluation. Tissues are processed inthe V.I.P. 2000 (Miles, Inc., Elkhart, Ind.) resulting in Paraffin®infiltration of the tissue. The tissue/Paraffin® blocks are sliced into5 μm sections in a Jung Biocut (Leica, Nussloch, Germany), placed onglass slides, and incubated at 60° C. for one hour to aid in adheringthe tissue to the slide. The Paraffin® is removed by ishing the slidesthree times in 100% xylene for 5 minutes. The slides are then rehydratedby 2 ishes in 100% ethanol for 3 minutes; followed by one ish in 95%ethanol. The slides are allowed to dry and then mounted with 5 to 10 μlof antifade medium which is prepared by adding nine parts glycerolcontaining 2% DABCO (1,4-diazobicyclo-(2,2,2,)-octane, Sigma, St. Louis,Mo.), dissolved at 55-70° C. to one part 0.2 M Tris/HCL, pH 7.5 DAPI(Sigma, St. Louis, Mo.) or propididum iodide (0.5 μg/ml). See alsoKievits, T. et al., Cytogenet Cell Cenet 53:134-136 (1990) for antifademedium. Slides are covered with cover slips and immediately examined byfluorescent microscopy at 495 nm.

Results are positive if the labeled ZS33LP shows increased fluorescencecompared to the glycine and “scrambled” controls.

Example 11 Gastric Contractility

Two male Sprague-Dawley rats, approximately 12 weeks old (Harlan,Indianapolis, Ind.) are anesthetized with urethane and their stomachsare exposed through a small abdominal incision. Two 2.4 mm transducingcrystals (Sonometrics, Ontario, Canada) are placed on the antral portionof the stomach such that circular contractions could be monitored as achange in the distance between the two crystals. The crystals areattached with VETBOND TISSUE ADHESIVE (3M, St. Paul, Minn.).

10 μl of 1 μM acetylcholine is applied topically to the stomach betweenthe two crystals, and results in a rapid, but transient increase in thedistance between two crystals. 10 μl of norepinephrine (NE) at 1 μMcauses a reduction in the distance between the two crystals. Theamplitude of the NE-induced decrease is approximately 50% of theacetylcholine-induced increase in distance. Both responses aretransient.

A negative control of 10 μl of phosphate buffer solution (PBS) appliedtopically between the crystals has no effect.

A peptide corresponding to the amino acid sequence of SEQ ID NOs:4, 7,10, 14, or 17 is dissolved in PBS) and 10 μl is applied topically for afinal concentration of 1 μg, 10 μg or 100 μg. The ZS33LP at 1 μg inducesa sustained, rhythmic increase and decrease in crystal distance.

Example 12 In Vivo Glucose Absorption

Eight female ob/ob mice, approximately 6 weeks old (Jackson Labs, BarHarbor, Me.) are adapted to a 4 hour daily feeding schedule for twoweeks. After two weeks on the feeding schedule, the mice are give 100 μgof a peptide corresponding to the amino acid sequence as shown in SEQ IDNOs:4-6, 9-11, 14-17, 20-22, or 25-26 in 100 μl sterile 0.1% BSA by oralgavage, immediately after their eating period (post-prandially). Thirtyminutes later, the mice are challenged orally with a 0.5 ml volume of25% glucose. Retroorbital bleeds are done to determine serum glucoselevels. Blood is drawn prior to peptide dosing, prior to oral glucosechallenge, and at 1, 2, 4, and 20 hours following the glucose challenge.

When zsig33-beta, zsig33-gamma, zsig33-delta or zsig33-epsilon peptideis given orally at 100 μg, 30 minutes prior to an oral glucosechallenge, an enhanced post-prandial glucose absorption is seen.

Example 13 Gastric Emptying

The effect of topically applied ZS33LP peptide (i.e. SEQ ID NOs:4-6,9-11, 14-17, 20-22, and 25-26) on the transit of phenol red through thestomachs of fasted male Sprague-Dawley rats (Harlan, Indianapolis, Ind.)is evaluated. The rats (6 animals, 8 weeks old) are fasted 24 hrs priorto being anesthetized with urethane (0.5 ml/100 grams of 25% solution).After anesthetizing, the animals are orally gavaged with 1 ml of PhenolRed solution (50 mg/ml in 2% methylcellulose solution).

The stomach of each animal is exposed through a small abdominal incisionand a ZS33LP or a 14 amino acid control of a scrambled sequence peptideis applied topically to the stomach five minutes following the gavage.The amount of Phenol Red remaining in the stomach is determined bymeasuring optical density of the extracted stomach contents 30 minutesafter the gavage.

Reduction in the amount of Phenol Red remaining in the stomach byapproximately 25% compared to a scrambled peptide, indicates that theZS33LP enhances gastric emptying in these rats.

Example 14 In Vitro Binding Assay

In vitro binding of ZS33LP polypeptides is performed similar to a methoddescribed by Miller, P., et al., Peptides 16:11-18, 1995. MaleSprague-Dawley rats, approximately 12 weeks old (Harlan, Indianapolis,Ind.) are sacrificed by IV pentobarbital injections. Tissue from theantrum is removed and the mucosal and serosal layers are dissected awayfor the smooth muscle. Tissue is minced and homogenized (BrinkmanPolytron, Luzern Switzerland), set at 10 for 60 seconds in 10 volumes ofTris-EDTA. The homogenate is centrifuged (1000×g for 15 minutes) and thesupernatant is discarded. Pellets are ished and passed through a nylonmesh to remove tissue clumps. Membrane protein is quantified.

ZS33LP polypeptides (corresponding to SEQ ID NOs:4-6, 9-11, 14-17,20-22, and 25-26) are iodinated by the chloramines-T method (See Boivin;M., et al., J. Gastrointest. Motil. 2:240-246, 1990). Iodinated peptidesare purified by HPLC. Binding of ¹²⁵ZS33LP on membrane extracts isperformed in a volume of 890 μl of 50 μM Tris-HCl (pH8.00), 1 mM EDTA,10 mM MgCl₂, and 2% BSA. The reaction is stopped by addition of 3 mlice-cold Tris-EDTA buffer. Free and bound counts are separated bycentrifugation at 1600×g for 10 minutes. Pellets are ished with 2.0 mlice-cold Tris-EDTA buffer, and centrifuged again. Radioactivity if thepellet is determined by a gamma counter. Specific binding is calculatedfrom total and nonspecific binding, determined in the absence andpresence of 10⁻⁵ cold ZS33LP.

Example 15 Effects of ZS33LP on Body Weight, Food Intake, and GlucoseClearance

Female ob/ob mice, 8 weeks old, (Jackson Labs, Bar Harbor, Me.) areadapted to a special 4 hour daily feeding schedule for two weeks. Theyare fed ad libitum from 7:30-11:30 am daily. After two weeks on thefeeding schedule, the mice are divided into six groups. Each group isgiven 1.0 mg/mouse of a ZS33LP (i.e., SEQ ID NO: 4, 7, 10, 14, or 17 ora scrambled sequence peptide for the negative control, in 100 ml sterile0.1% BSQA by oral gavage just prior to receiving food, and, again at theend of the 4 hour feeding period. The mice are injected twice daily forfourteen days, during which time food intake and body weight is measureddaily. On day 14, immediately after the second oral gavage of thezsig33-1 peptide, the mice are challenged orally with an 0.5 ml volumeof 25% glucose. Retro-orbital bleeds are done to determine serum glucoselevels immediately prior to administration of the ZS33LP or vehicle(t=30 min.), and also at 0, 1, 2, and 4 hours following the glucosechallenge.

The effect on daily body weight or food intake measurements, or onglucose clearance is determined on day 14.

Example 16 Summary of Peptide Synthesis

Zsig33-like peptides corresponding to SEQ ID NOs: 4, 11, 14, 20 and 26were synthesized with Fmoc chemistry on a model 431A Peptide Synthesizer(Applied Biosystems, Foster City, Calif.). Fmoc-Amide resin (AppliedBiosystems) was used as the initial support resin for the zsig33-epsilonand zsig33-beta peptides. Fmoc-Lys(Boc) Wang resin (Anaspec, San Jose,Calif.) was used for the zsig33-delta and zsig33-gamma peptides.Fmoc-Arg(Pbf) Wang resin (Anaspec) was used for the zsig33-linker. Aminoacid cartridges were obtained from Anaspec. A mixture of2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyluronium hexafluorophosphate(HBTU), 1-Hydroxybenzotriazole (HOBt), N,N-Diisopropylethylamine,N-Methylpyrrolidone, Dichloromethane (all from Applied Biosystems) andPiperidine (Aldrich Chemical Co., Milwaukee, Wis.), were used assynthesis reagents. The peptides were cleaved from the solid supportwith 95% TFA (J. T. Baker, Phillipsburg, N.J.). Purification of thepeptides were done by RP-HPLC using a C18, 10 micron, 22×250 mmsemi-preparative column (Vydac, Hesperia, CA). Eluted fractions from thecolumn were collected and analyzed for correct mass and purity byelectrospray mass spectrometry and by analytical RP-HPLC. Purifiedpeptides were lyophilized to dryness.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide encoding a polypeptide, wherein the polypeptide consists of an amino acid sequence selected from the group consisting of: a) the amino acid sequence of SEQ ID NO:9; b) the amino acid sequence of SEQ ID NO:10; and c) the amino acid sequence of SEQ ID NO:11.
 2. The isolated polynucleotide according to claim 1, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence of SEQ ID NO:9.
 3. The isolated polynucleotide according to claim 2, wherein the polypeptide has an addition, and wherein the addition is selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 4. The isolated polynucleotide according to claim 3, wherein the amino-terminal extension is an amino-terminal methionine.
 5. The isolated polynucleotide according to claim 3, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine.
 6. An isolated polynucleotide consisting of residues 275 to 349 of SEQ ID NO:
 1. 7. The isolated polynucleotide according to claim 6, wherein the polynucleotide encodes a polypeptide and wherein the polypeptide has an addition selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 8. The isolated polynucleotide according to claim 7, wherein the amino-terminal extension is an amino-terminal methionine.
 9. The isolated polynucleotide according to claim 7, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine.
 10. The isolated polynucleotide according to claim 1, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence of SEQ ID NO:10.
 11. The isolated polynucleotide according to claim 10, wherein the polypeptide has an addition, and wherein the addition is selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 12. The isolated polynucleotide according to claim 11, wherein the amino-terminal extension is an amino-terminal methionine.
 13. The isolated polynucleotide according to claim 11, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine.
 14. An isolated polynucleotide consisting of residues 275 to 346 of SEQ ID NO:
 1. 15. The isolated polynucleotide according to claim 14, wherein the polynucleotide encodes a polypeptide and wherein the polypeptide has an addition selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 16. The isolated polynucleotide according to claim 15, wherein the amino-terminal extension is an amino-terminal methionine.
 17. The isolated polynucleotide according to claim 15, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine.
 18. The isolated polynucleotide according to claim 1, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence of SEQ ID NO:11.
 19. The isolated polynucleotide according to claim 18, wherein the polypeptide has an addition, and wherein the addition is selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 20. The isolated polynucleotide according to claim 19, wherein the amino-terminal extension is an amino-terminal methionine.
 21. The isolated polynucleotide according to claim 19, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine.
 22. An isolated polynucleotide consisting of residues 275 to 343 of SEQ ID NO:
 1. 23. The isolated polynucleotide according to claim 22, wherein the polynucleotide encodes a polypeptide and wherein the polypeptide has an addition selected from the group consisting of: a) an amino-terminal extension; b) a carboxyl-terminal extension; c) a linker peptide; and d) an affinity tag.
 24. The isolated polynucleotide according to claim 23, wherein the amino-terminal extension is an amino-terminal methionine.
 25. The isolated polynucleotide according to claim 23, wherein the amino-terminal extension or the carboxyl-terminal extension is a cysteine. 